CA2928078A1 - Lipid formulations for delivery of messenger rna - Google Patents

Lipid formulations for delivery of messenger rna Download PDF

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Publication number
CA2928078A1
CA2928078A1 CA2928078A CA2928078A CA2928078A1 CA 2928078 A1 CA2928078 A1 CA 2928078A1 CA 2928078 A CA2928078 A CA 2928078A CA 2928078 A CA2928078 A CA 2928078A CA 2928078 A1 CA2928078 A1 CA 2928078A1
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mos
siuoultpoqulo
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Michael Heartlein
Daniel Anderson
Yizhou Dong
Frank Derosa
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Massachusetts Institute of Technology
Translate Bio Inc
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Shire Human Genetics Therapies Inc
Massachusetts Institute of Technology
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Abstract

The present invention provides, among other things, methods of delivering mRNA in vivo, including administering to a subject in need of delivery a composition comprising an mRNA encoding a protein, encapsulated within a liposome such that the administering of the composition results in the expression of the protein encoded by the mRNA in vivo, wherein the liposome comprises a cationic lipid of formula I-c: or a pharmaceutically acceptable salt thereof.

Description

LIPID FORMULATIONS FOR DELIVERY OF MESSENGER RNA
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application Serial No.
61/894,299, filed October 22, 2013 and U.S. Provisional Application Serial No.
61/953,516, filed March 14, 2014, the disclosures of which are hereby incorporated by reference.
SEQUENCE LISTING
[0002] The present specification makes reference to a Sequence Listing (submitted electronically as a .txt file named "2006685-0687 SL.txt" on October 22, 2014). The .txt file was generated on October 22, 2014 and is 35,552 bytes in size. The entire contents of the Sequence Listing are herein incorporated by reference.
BACKGROUND
[0003] Delivery of nucleic acids has been explored extensively as a potential therapeutic option for certain disease states. In particular, RNA interference (RNAi) has been the subject of significant research and clinical development. While RNAi, such as short interfering RNA
(siRNA), may have therapeutic potential, it is of little use in treating diseases involving deficiency of one or more proteins. messenger RNA (mRNA) therapy has become an increasingly important option for treatment of various diseases, in particular, for those associated with deficiency of one or more proteins.
SUMMARY OF THE INVENTION
[0004] The present invention provides improved methods and compositions for highly efficient delivery and expression of mRNA and encoded protein in vivo. The invention is based, in part, on the surprising discovery that liposomes based on a particular class of cationic lipids, such as, those having a structure of formula I-c described herein, are unexpectedly effective in delivering mRNA and producing encoded protein in vivo, more effective even as compared to those cationic lipids that were considered to be among the best in delivering mRNA in the prior art. Indeed, prior to the present invention, cationic lipids have been extensively explored as an important component of liposomes typically used to encapsulate nucleic acids including mRNA
for in vivo delivery. Due to the uniquely fragile and long structure of mRNA
and the complicated in vivo translation process, cationic lipids used in the liposomes typically play two roles. First, cationic lipids promote interaction with negatively charged mRNA
during encapsulation, circulation and endocytosis, thereby capturing and protecting the mRNA. Then, once inside cytosol, cationic lipids need to be able to release the mRNA so that the mRNA can be translated to produce encoded protein. Some cationic lipids, in particular, those known as titratable cationic lipids are particularly effective in delivering mRNA. One example of such cationic lipids known to be capable of efficient delivery of mRNA is C12-200.
Surprisingly, the present inventors found that cationic lipids described herein can be even more effective in delivering various mRNA in vivo, than those best known in the prior art including C12-200. For example, as shown in the Examples below, liposome particles incorporating a cationic lipid described herein (e.g., cKK-E12) resulted in close to 50% higher protein expression of human Factor IX protein detected in the plasma of administered mice, as compared to C12-200-based liposome particles. Furthermore, the plasma residence time of different proteins expressed from mRNA delivered by cKK-E12 based liposomes is sustained up to 7 days or longer post a single administration. Thus, the present inventors have demonstrated that this class of cationic lipids having a structure of formula I-c described herein (e.g., cKK-E12) can be uniquely useful in delivering mRNA for highly efficient and sustained production of protein (e.g., therapeutic protein) in vivo. The present invention therefore permits an improved mRNA
therapy that can significantly reduce required amount of mRNA and associated lipids, administration frequency, and possible side effects, providing more potent, safer, and patient friendly mRNA therapy for various diseases.
[0005] In one aspect, the present invention provides methods of delivering messenger RNA (mRNA) in vivo, including administering to a subject in need of delivery a composition comprising an mRNA encoding a protein, encapsulated within a liposome such that the administering of the composition results in the expression of the protein encoded by the mRNA
in vivo, wherein the liposome comprises a cationic lipid of formula I-c:

R6õR7 N
R2 - II_ -\
N

R6\ N

I-C
or a pharmaceutically acceptable salt thereof, wherein:
p is an integer of between 1 and 9, inclusive;
each instance of R2 is independently hydrogen or optionally substituted C1_6 alkyl;
each instance of R6 and R7 is independently a group of the formula (i), (ii), or (iii);
Formulae (i), (ii), and (iii) are:
XRL
RI-)--YRP µ

RL
R' 5 Or (0 (ii) (iii) wherein:
each instance of R' is independently hydrogen or optionally substituted alkyl;
X is 0, S, or NRx, wherein Rx is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;

Y is 0, S, or NR, wherein RY is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;
RP is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom; and RI- is optionally substituted C1_50 alkyl, optionally substituted C2_50 alkenyl, optionally substituted C2_50 alkynyl, optionally substituted heteroC1_50 alkyl, optionally substituted heteroC2_50 alkenyl, optionally substituted heteroC2_50 alkynyl, or a polymer.
[0006] In another aspect, the present invention provides methods of treating a disease or disorder including administering to subject in need of treatment a composition comprising an mRNA encoding a therapeutic protein encapsulated within a liposome such that the administering of the composition results in the expression of the protein encoded by the mRNA
in one or more tissues affected by the disease or disorder, wherein the liposome comprises a cationic lipid having a structure of formula I-c.
[0007] In another aspect, the present invention provides compositions for delivery of messenger RNA (mRNA) comprising an mRNA encoding a protein encapsulated within a liposome, wherein the liposome comprises a cationic lipid having a structure of formula I-c.
[0008] In some embodiments, a suitable cationic lipid is cKK-E12:

HO
¨(CH2)9CH3 7 ________________________________________________ _,...
(cH2)9cH3 /
HO
HN
CI¨NH
OH
H3C(H2C)9 ______________ K._ __ /
/
N
HO¨

(CH2)9CH3
[0009] In some embodiments, a suitable liposome further comprises one or more non-cationic lipids, one or more cholesterol-based lipids and/or one or more PEG-modified lipids. In some embodiments, the one or more non-cationic lipids are selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-0-monomethyl PE, 16-0-dimethyl PE, 18-1-trans PE, 1-stearoy1-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixture thereof
[0010] In some embodiments, a suitable liposome further comprises one or more cholesterol-based lipids. In some embodiments, the one or more cholesterol-based lipids are selected from cholesterol, PEGylated cholesterol and DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine.
[0011] In some embodiments, a suitable liposome further comprises one or more PEG-modified lipids. In some embodiments, the one or more PEG-modified lipids comprise a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. In some embodiments, a PEG-modified lipid is a derivatized ceramide such as N-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000].
In some embodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterol or Dimyristoylglycerol (DMG) -PEG-2K.
[0012] In some embodiments, a suitable liposome comprises cKK-E12, DOPE, cholesterol and DMG-PEG2K.
[0013] In some embodiments, the cationic lipid (e.g., cKK-E12) constitutes about 30-50 % (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by molar ratio. In some embodiments, the cationic lipid (e.g., cKK-E12) constitutes about 30%, about 35%, about 40 %, about 45%, or about 50% of the liposome by molar ratio.
[0014] In particular embodiments, the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:30:20:10 by molar ratio. In particular embodiments, the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:30:25:5 by molar ratio.
In particular embodiments, the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:32:25:3 by molar ratio.
[0015] In some embodiments, a suitable liposome has a size of or less than about 500nm, 450 nm, 400nm, 350 nm, 300nm, 250 nm, 200 nm, 150 nm, 125 nm, 110 nm, 100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, or 50 nm.
[0016] In some embodiments, a composition according to the invention is administered intravenously. In some embodiments, a composition according to the invention is administered via pulmonary delivery. In some embodiments, the pulmonary delivery is by aerosolization, inhalation, nebulization or instillation. In some embodiments, a composition according to the invention is administered intrathecally. In some embodiments, the composition is formulated as respirable particles, nebulizable lipid, or inhalable dry powder.
[0017] In some embodiments, the expression of the protein encoded by the mRNA is detectable in liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and/or cerebrospinal fluid.
[0018] In some embodiments, the expression of the protein encoded by the mRNA is detectable 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and/or 72 hours after the administration. In some embodiments, the expression of the protein encoded by the mRNA is detectable 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and/or 7 days after the administration.
In some embodiments, the expression of the protein encoded by the mRNA is detectable 1 week, 2 weeks, 3 weeks, and/or 4 weeks after the administration. In some embodiments, the expression of the protein encoded by the mRNA is detectable after a month after the administration.
[0019] In some embodiments, the protein encoded by the mRNA is a cytosolic protein.
In some embodiments, the protein encoded by the mRNA is a secreted protein. In some embodiments, the protein encoded by the mRNA is an enzyme. In some embodiments, the mRNA has a length of or greater than about 0.5kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb. In some embodiments, the protein encoded by the mRNA is Argininosuccinate Synthetase (ASS1), Factor IX, survival of motor neuron 1, or phenylalanine hydroxylase.
[0020] In some embodiments, the mRNA is administered at a dose ranging from about 0.1 - 5.0 mg /kg body weight, for example about 0.1 -4.5, 0.1 -4.0, 0.1 -3.5, 0.1 -3.0, 0.1 -2.5, 0.1 -2.0, 0.1 - 1.5, 0.1 - 1.0, 0.1 -0.5, 0.1 -0.3, 0.3 -5.0, 0.3 -4.5, 0.3 - 4.0, 0.3 - 3.5, 0.3 - 3.0, 0.3 -2.5, 0.3 -2.0, 0.3 - 1.5, 0.3 - 1.0, 0.3 - 0.5, 0.5 -5.0, 0.5-4.5, 0.5 -4.0, 0.5 -3.5, 0.5 -3.0,0.5-2.5,0.5-2.0,0.5-1.5, or 0.5 - 1.0 mg/kg body weight. In some embodiments, the mRNA is administered at a dose of or less than about 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.8, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mg/kg body weight.
[0021] In some embodiments, the mRNA comprises one or more modified nucleotides.
In some embodiments, the one or more modified nucleotides comprise pseudouridine, N-1-methyl-pseudouridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and/or 2-thiocytidine. In some embodiments, the mRNA is unmodified.
[0022] Other features, objects, and advantages of the present invention are apparent in the detailed description, drawings and claims that follow. It should be understood, however, that the detailed description, the drawings, and the claims, while indicating embodiments of the present invention, are given by way of illustration only, not limitation.
Various changes and modifications within the scope of the invention will become apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWING
[0023] The drawings are for illustration purposes only not for limitation.
[0024] Figure 1 shows an exemplary graph of the levels of human factor IX
(FIX) detected in the serum of treated mice 24 hours after administration of C12-200 or cKK-E12 liposomes containing FIX mRNA.
[0025] Figure 2 shows an exemplary graph of FIX detected in the plasma of mice treated with 0.1, 0.3, 0.6, 1.0, or 3.0 mg/kg of one of two ratios of FIX mRNA
containing cKK-E12 liposomes either 6 or 24 hours after administration.
[0026] Figure 3 shows an exemplary graph of the level of ASS1 protein detected in the livers of mice treated with 0.1, 0.3, 0.6, 1.0, or 3.0 mg/kg of ASS1 mRNA-containing cKK-E12 liposomes 24 hours after administration.
[0027] Figure 4 shows exemplary western blot analyses of ASS1 protein levels in the liver 24 hours post administration of 0.1, 0.3, 0.6, 1.0, or 3.0 mg/kg of cKK-E12 liposomes containing ASS1 mRNA.
[0028] Figure 5 shows an exemplary graph of ASS1 protein levels in the liver of mice 0.5, 3, 6, 12, 24, 48, 72 hours after a single IV injection of ASS1 mRNA
containing cKK-E12 liposomes (lmg/kg). Also shown is the level of ASS1 protein 7 days after administration.
[0029] Figure 6 shows exemplary western blot analyses of ASS1 protein levels in the liver 0.5, 3, 6, 12, 24, 48, 72 hours after a single IV injection of lmg/kg ASS1 mRNA containing cKK-E12 liposomes. Also shown is the level of ASS1 protein 7 days after administration.
[0030] Figure 7 ¨ shows detection of human ASS1 messenger RNA via in situ hybridization in the livers of treated mice. Exogenous mRNA is observable for at least 72 hr post-administration after a single dose (1.0 mg/kg) of ASS1 mRNA-loaded MD1-based lipid nanoparticles. Human ASS1 mRNA is detectable in sinusoidal cells as well as hepatocytes.
[0031] Figure 8 ¨ shows exemplary immunohistochemical staining of ASS1 protein levels in mouse liver 24 hours after administration of 1 mg/kg ASS1 mRNA
containing cKK-E12 lipid nanoparticles. Human ASS1 protein is detectable in sinusoidal cells as well as hepatocytes.
[0032] Figure 9 shows low magnification (4x) immunohistochemical staining of ASS1 protein levels in mouse liver 24 hours after administration of 1 mg/kg ASS1 mRNA containing cKK-E12 liposomes. A comparison to untreated mouse liver (left) demonstrates the widespread distribution of human ASS1 protein throughout the liver.
[0033] Figure 10 shows exemplary results illustrating that cKK-E12 lipid nanoparticles efficiently delivered FL mRNA via nebulization. Mice were exposed to milligram of encapsulated FL mRNA and analysis was performed 24 hours post-exposure.
[0034] Figure 11 illustrates detection via western blot of human SMN-1 protein derived from exogenous hSMN-1 mRNA that was transfected into BHK-21 cells. Various antibodies specific to human SMN were employed: (A) anti-SMN 4F11 antibody at 1:1,000 dilution; (B) Pierce PA5-27309 a-SMN antibody at 1:10,000 dilution; and (C) LSBio C138149 a-SMN
antibody at 1:10,000 dilution.
[0035] Figure 12A-C illustrates multiplex nucleic acid in situ detection of human Survival of Motor Neuron (hSMN-1) mRNA in (A) Cervical, (B) Thoracic and (C) Lumbar spinal tissue, 24 hours post intrathecal delivery.
[0036] Figure 13 illustrates positive detection of human SMN-1 protein produced in the spinal cord of a rat 24 hours post-intrathecal administration of human SMN-1 mRNA-loaded lipid nanoparticles. Anti-human SMN 4F11 antibody was employed at 1:2500 dilution. Panel A
represents treated rat spinal cord tissue and panel B represents untreated rat spinal cord tissue.
[0037] Figure 14 In vivo transfection of CFTR knockout mice with C-terminal Hisio tagged (SEQ ID NO: 11) codon-optimized human CFTR mRNA encapsulated within either a lipid (cKK-E12) or polymeric (PEI) nanoparticle formulation. Following nebulized delivery of each respective mRNA formulation, Right and Left lung tissue lysate was collected and analyzed for CFTR expression by Western blot using anti-His antibody 1187. Control CFTR
knockout lung tissue and CFTR-Hisio HEK293 lysate ("Hisio" disclosed as SEQ ID NO: 11) was used as a negative and positive controls respectively.
[0038] Figure 15 illustrates positive detection of active firefly luciferase (FFL) protein in a treated pig lung via luminescence upon exposure to FFL/CO-CFTR-C-Hisio mRNA
("Hisio"
disclosed as SEQ ID NO: 11) encapsulated cKK-E12 lipid nanoparticles. Pigs were treated with 1 mg FFL + 9 mg CO-CFTR-C-Hisio mRNA ("Hisio" disclosed as SEQ ID NO: 11) encapsulated lipid nanoparticles via nebulization using a Pan i jet nebulizer and sacrificed 24 hours post-treatment. FFL luminescence was visualized using an IVIS
bioluminometer.
DEFINITIONS
[0039] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.
[0040] Amino acid: As used herein, term "amino acid," in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure HEN¨C(H)(R)¨COHO. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is an 1-amino acid. "Standard amino acid"
refers to any of the twenty standard 1-amino acids commonly found in naturally occurring peptides.
"Nonstandard amino acid" refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions. Amino acids, including carboxyl- and/or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, protecting groups, and/or substitution with other chemical groups that can change the peptide's circulating half-life without adversely affecting their activity. Amino acids may participate in a disulfide bond. Amino acids may comprise one or posttranslational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.). The term "amino acid" is used interchangeably with "amino acid residue," and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.
[0041] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans, at any stage of development. In some embodiments, "animal" refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms.
In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.
[0042] Approximately or about: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
[0043] Delivery: As used herein, the term "delivery" encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations in which an mRNA
is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (aslo referred to as "local distribution" or "local delivery"), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery).
[0044] Expression: As used herein, "expression" of a nucleic acid sequence refers to translation of an mRNA into a polypeptide, assemble multiple polypeptides (e.g., heavy chain or light chain of antibody) into an intact protein (e.g., antibody) and/or post-translational modification of a polypeptide or fully assembled protein (e.g., antibody). In this application, the terms "expression" and "production," and grammatical equivalent, are used inter-changeably.
[0045] Functional: As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
[0046] Half-life: As used herein, the term "half-life" is the time required for a quantity such as nucleic acid or protein concentration or activity to fall to half of its value as measured at the beginning of a time period.
[0047] Improve, increase, or reduce: As used herein, the terms "improve,"
"increase" or "reduce," or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein. A "control subject" is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.
[0048] In Vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
[0049] In Vivo: As used herein, the term "in vivo" refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
[0050] Isolated: As used herein, the term "isolated" refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, calculation of percent purity of isolated substances and/or entities should not include excipients (e.g., buffer, solvent, water, etc.).
[0051] Local distribution or delivery: As used herein, the terms "local distribution,"
"local delivery," or grammatical equivalent, refer to tissue specific delivery or distribution.
Typically, local distribution or delivery requires a protein (e.g., enzyme) encoded by mRNAs be translated and expressed intracellularly or with limited secretion that avoids entering the patient's circulation system.
[0052] messenger RNA (mRNA): As used herein, the term "messenger RNA
(mRNA)"
refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA
sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine);
nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine);
chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).
[0053] In some embodiments, the mRNA comprises one or more nonstandard nucleotide residues. The nonstandard nucleotide residues may include, e.g., 5-methyl-cytidine ("5mC"), pseudouridine ("yU"), and/or 2-thio-uridine ("2sU"). See, e.g., U.S. Patent No. 8,278,036 or W02011012316 for a discussion of such residues and their incorporation into mRNA. The mRNA may be RNA, which is defined as RNA in which 25% of U residues are 2-thio-uridine and 25% of C residues are 5-methylcytidine. Teachings for the use of RNA are disclosed US
Patent Publication U520120195936 and internation publication W02011012316, both of which are hereby incorporated by reference in their entirety. The presence of nonstandard nucleotide residues may render an mRNA more stable and/or less immunogenic than a control mRNA with the same sequence but containing only standard residues. In further embodiments, the mRNA
may comprise one or more nonstandard nucleotide residues chosen from isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine and 2-chloro-6-aminopurine cytosine, as well as combinations of these modifications and other nucleobase modifications. Certain embodiments may further include additional modifications to the furanose ring or nucleobase. Additional modifications may include, for example, sugar modifications or substitutions (e.g., one or more of a 2'-0-alkyl modification, a locked nucleic acid (LNA)). In some embodiments, the RNAs may be complexed or hybridized with additional polynucleotides and/or peptide polynucleotides (PNA).
In embodiments where the sugar modification is a 2'-0-alkyl modification, such modification may include, but are not limited to a 2'-deoxy-2'-fluoro modification, a 2'-0-methyl modification, a 2'-0-methoxyethyl modification and a 2'-deoxy modification. In certain embodiments, any of these modifications may be present in 0-100% of the nucleotides¨for example, more than 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100% of the constituent nucleotides individually or in combination.
[0054] Nucleic acid: As used herein, the term "nucleic acid," in its broadest sense, refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, "nucleic acid" refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" encompasses RNA as well as single and/or double-stranded DNA and/or cDNA.
[0055] Patient: As used herein, the term "patient" or "subject" refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. A human includes pre and post natal forms.
[0056] Pharmaceutically acceptable: The term "pharmaceutically acceptable"
as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[0057] Systemic distribution or delivery: As used herein, the terms "systemic distribution," "systemic delivery," or grammatical equivalent, refer to a delivery or distribution mechanism or approach that affect the entire body or an entire organism.
Typically, systemic distribution or delivery is accomplished via body's circulation system, e.g., blood stream.
Compared to the definition of "local distribution or delivery."
[0058] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term "subject" is used herein interchangeably with "individual" or "patient." A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
[0059] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
[0060] Target tissues: As used herein , the term "target tissues" refers to any tissue that is affected by a disease to be treated. In some embodiments, target tissues include those tissues that display disease-associated pathology, symptom, or feature.
[0061] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.
[0062] Treating: As used herein, the term "treat," "treatment," or "treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.
DETAILED DESCRIPTION
[0063] The present invention provides, among other things, methods and compositions for delivering mRNA in vivo using improved liposomes incorporating cationic lipids described herein.
Liposomes for mRNA Delivery
[0064] As used herein, the term "liposome" refers to any lamellar, multilamellar, or solid lipid nanoparticle vesicle. Typically, a liposome as used herein can be formed by mixing one or more lipids or by mixing one or more lipids and polymer(s). Thus, the term "liposome" as used herein encompasses both lipid and polymer based nanoparticles. In particular, a liposome according to the present invention incorporates a cationic lipid described herein. As a non-limiting example, a cationic lipid suitable for the present invention is cKK-E12, or (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione), as described in more detail below. A
suitable liposome may also contain second or additional cationic lipids, helper lipids (e.g., non-cationic lipids and/or cholesterol-based lipids), PEG-modified lipids, and/or polymers.
[0065] In some embodiments, cationic lipid(s) (e.g., cKK-E12) constitute(s) about 30-50 % (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by molar ratio. In some embodiments, the cationic lipid (e.g., cKK-E12) constitutes about 30%, about 35%, about 40 %, about 45%, or about 50% of the liposome by molar ratio.
Cationic Lipids
[0066] In some embodiments, provided liposomes or compositions provided comprise a cationic lipid according to formula I:
¨ ¨
R\2 R1 N
Q- --Q
N

_ - P
, I
or a pharmaceutically acceptable salt thereof, wherein:
p is an integer of between 1 and 9, inclusive;
each instance of Q is independently 0, S, or NRQ;
RQ is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted
67 PCT/US2014/061793 heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group, or a group of the formula (i), (ii) or (iii);
each instance of R1 is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, halogen, ¨ORA1, _N(RA)2, ¨SRA1, or a group of formula (iv):

(iv) L is an optionally substituted,alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, or optionally substituted heteroarylene, or combination thereof, and each of R6 and R7 is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group, or a group of formula (i), (ii) or (iii);
each occurrence of RA1 is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to an sulfur atom, a nitrogen protecting group when attached to a nitrogen atom, or two RA1 groups, together with the nitrogen atom to which they are attached, are joined to form an optionally substituted heterocyclic or optionally substituted heteroaryl ring;

each instance of R2 is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group, or a group of the formula (i), (ii), or (iii);
Formulae (i), (ii), and (iii) are:
R' XRL
R'\

K
RL
- R' Ri -1-1 R' (i) (ii) , (iii) , each instance of R' is independently hydrogen or optionally substituted alkyl;

Xis 0, S, or NRx;
Rx is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;
Y is 0, S, or NR';
RY is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;
RP is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom;
RI- is optionally substituted C1-50 alkyl, optionally substituted C2_50 alkenyl, optionally substituted C2_50 alkynyl, optionally substituted heteroC1_50 alkyl, optionally substituted heteroC2_50 alkenyl, optionally substituted heteroC2_50 alkynyl, or a polymer;
provided that at least one instance of RQ, R2, R6, or R7 is a group of the formula (i), (ii), or (iii).
[0067] In some embodiments, a cationic lipid in a provided composition or method is a compound of formula I. In some embodiments, a cationic lipid in a provided composition or method is a compound of formula I, wherein the compound comprises one or more basic groups.
In some embodiments, a cationic lipid in a provided composition or method is a compound of formula I, wherein the compound comprises one or more amino groups.
[0068] In certain embodiments, a group of formula (i) represents a group of formula (i-a) or a group of formula (i-b):
RL R R' YRP R'\ YRP
_1 R' RL
(i-a) (i-b) wherein each variable is independently as defined above and described herein.
In some embodiments, a group of formula (i) is a group of formula (i-a). In some embodiments, a group of formula (i) is a group of formula (i-b).
[0069] In some embodiments, at least one instance of Rl is a group of formula (iv). In some embodiments, at least one instance of Rl is a group of formula (iv), wherein at least one of R6 and R7 is a group of formula (i), (ii) or (iii). In some embodiments, at least one instance of Rl is a group of formula (iv), wherein each of R6 and R7 is independently a group of formula (i), (ii) or (iii).
[0070] In some embodiments, each Rl is independently a group of formula (iv). In some embodiments, each Rl is independently a group of formula (iv), wherein at least one of R6 and R7 is a group of formula (i), (ii) or (iii). In some embodiments, each Rl is independently a group of formula (iv), wherein each of R6 and R7 is independently a group of formula (i), (ii) or (iii). In some embodiments, each Rl is independently a group of formula (iv), wherein each of R6 and R7 is independently a group of formula (i). In some embodiments, each Rl is independently a group of formula (iv), wherein each of R6 and R7 is independently a group of formula (ii). In some embodiments, each Rl is independently a group of formula (iv), wherein each of R6 and R7 is independently a group of formula (iii). In some embodiments, each Rl is independently a group of formula (iv), wherein each of R6 and R7 is independently a group of formula (i-a). In some embodiments, each Rl is independently a group of formula (iv), wherein each of R6 and R7 is independently a group of formula (i-b).
[0071] In some embodiments, each instance of R' is hydrogen.
[0072] In some embodiments, L is an optionally substituted alkylene.
ir
[0073] In some embodiments, a group of formula (iv) is of formula "(21 , wherein q is an integer between 1 and 50, inclusive, and each of R6 and R7 is independently as defined above and described herein.
[0074] As generally defined above, p is an integer of between 1 and 9, inclusive. In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3. In certain embodiments, p is 4. In certain embodiments, p is 5. In certain embodiments, p is 6. In certain embodiments, p is 7. In certain embodiments, p is 8. In certain embodiments, p is 9.
[0075] In some embodiments, p is 1. In some embodiments, a compound of formula I is a compound of formula (I-a):

N
Q-N --Q
N
R1 R2 , I-a or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.
[0076] In some embodiments, p is 2. In some embodiments, a compound of formula I is a compound of formula (I-p2):
-,, \N .................................. ( Q ie.
=, R1 N¨R 2 Q, I-p2 or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.
[0077] In some embodiments, p is 3. In some embodiments, a compound of formula I is a compound of formula (I-p3):

jt,Ri.....,, ...R2 N
RI
T, QxN a Kr.Q
RI N I\L`R2 i R2 RI , I-p3 or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.
[0078] In some embodiments, p is 4. In some embodiments, a compound of formula I is a compound of formula (I-p4):

N' N

I-p4 or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.
[0079] In some embodiments, p is 5. In some embodiments, a compound of formula I is a compound of formula (I-p5):

Q N2 R2, R2 ,N Ri R y ,R2 ,R2õ

I
QTõN

R Q
I-p5 or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.
[0080] In some embodiments, p is 6. In some embodiments, a compound of formula I is a compound of formula (I-p6):

RyLQ IL.,e N , x ,N

Q N-,R2 Q
y.

R2 \
I N
RI
Q, I-p6 or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.
[0081] In some embodiments, p is 7. In some embodiments, a compound of formula I is a compound of formula (I-p7):
Q 51 ik) R1 CLXN...., ..) R- R2-'N W

/Q

CnNIR2 Ri'N1------(LRI

Q W , I-p7 or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.
[0082] In some embodiments, p is 8. In some embodiments, a compound of formula I is a compound of formula (I-p8):

R --N, /2 _ N )-- 1 (.\\

R.-________________________________ R1 ,) 0 R2_,N

) __ .(' Q 0 N¨R2 Q fq---4, ><).
R2 ¨NR1 \ , , I-p8 or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.
[0083] In some embodiments, p is 9. In some embodiments, a compound of formula I is a compound of formula (I-p9):

Ryt., ,,,,Iy14,4yit,, ..FlyQ
N N
i 1 R2 2 R1 R22 õN R1 X R R y R2 R2..., .õ..
R/ N''' R2 R1 0 R2 N a i 1 N N
, , I-p9 or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.
[0084] As generally defined above, each instance of Q is independently 0, S, or NO, wherein RQ is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group, or a group of the formula (i), (ii), or (iii).
[0085] In certain embodiments, at least one instance of Q is 0. In certain embodiments, each instance of Q is 0. In certain embodiments, at least one instance of Q is S. In certain embodiments, each instance of Q is S. In certain embodiments, at least one instance of Q is NO, wherein RQ is as defined above and described herein. In certain embodiments, each instance of Q is NO, wherein each RQ is independently as defined above and described herein. In certain embodiments, each instance of RQ is independently hydrogen or a group of the formula (i), (ii), or (iii).
[0086] As generally defined above, RQ is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group, or a group of the formula (i), (ii) or (iii).
[0087] In some embodiments, RQ is hydrogen. In some embodiments, RQ is optionally substituted alkyl. In some embodiments, RQ is optionally substituted alkenyl.
In some embodiments, RQ is optionally substituted alkynyl. In some embodiments, RQ is carbocyclyl. In some embodiments, RQ is optionally substituted heterocyclyl. In some embodiments, RQ is optionally substituted aryl. In some embodiments, RQ is optionally substituted heteroaryl. In some embodiments, RQ is a nitrogen protecting group. In some embodiments, RQ
is a group of formula (i), (ii) or (iii). In some embodiments, RQ is a group of formula (i).
In some embodiments, RQ is a group of formula (ii). In some embodiments, RQ is a group of formula (iii).
[0088]1 i As generally defined above, each instance of R s independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, halogen, ¨OR", _N(RA)2, or ¨SRA1, or a group of formula (iv), wherein each of RA1 and formula (iv) is independently as defined above and described herein.
[0089] In some embodiments, Rl is hydrogen.
[0090] In certain embodiments, Rl is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. In certain embodiments, at least one instance of Rl is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl.
[0091] In certain embodiments, Rl is optionally substituted alkyl; e.g., optionally substituted Ci_6alkyl, optionally substituted C2_6a1ky1, optionally substituted C3_6a1ky1, optionally substituted C4_6a1ky1, optionally substituted C4_5a1ky1, or optionally substituted C3_4a1ky1. In certain embodiments, at least one instance of Rl is optionally substituted alkyl; e.g., optionally substituted Ci_6alkyl, optionally substituted C2_6a1ky1, optionally substituted C3_6a1ky1, optionally substituted C4_6a1ky1, optionally substituted C4_5a1ky1, or optionally substituted C3_4a1ky1.
[0092] In certain embodiments, Rl is optionally substituted alkenyl, e.g., optionally substituted C2_6alkenyl, optionally substituted C3_6alkenyl, optionally substituted C4_6alkenyl, optionally substituted C4_5alkenyl, or optionally substituted C3_4alkenyl. In certain embodiments, at least one instance of Rl is optionally substituted alkenyl, e.g., optionally substituted C2-6alkenyl, optionally substituted C3_6alkenyl, optionally substituted C4_6alkenyl, optionally substituted C4_5alkenyl, or optionally substituted C3_4alkenyl.
[0093] In certain embodiments, Rl is optionally substituted alkynyl, e.g., optionally substituted C2_6alkynyl, optionally substituted C3_6alkynyl, optionally substituted C4_6alkynyl, optionally substituted C4_5alkynyl, or optionally substituted C3_4alkynyl. In certain embodiments, at least one instance of Rl is optionally substituted alkynyl, e.g., optionally substituted C2_ 6alkynyl, optionally substituted C3_6alkynyl, optionally substituted C4_6alkynyl, optionally substituted C4_5alkynyl, or optionally substituted C3_4alkynyl.
[0094] In certain embodiments, Rl is optionally substituted carbocyclyl, e.g., optionally substituted C3_10 carbocyclyl, optionally substituted C5_8 carbocyclyl, optionally substituted C5_6 carbocyclyl, optionally substituted C5 carbocyclyl, or optionally substituted C6 carbocyclyl. In certain embodiments, at least one instance of Rl is optionally substituted carbocyclyl, e.g., optionally substituted C3_10 carbocyclyl, optionally substituted C5_8 carbocyclyl, optionally substituted C5_6 carbocyclyl, optionally substituted C5 carbocyclyl, or optionally substituted C6 carbocyclyl.
[0095] In some embodiments, Rl is optionally substituted heterocyclyl, e.g., optionally substituted 3-14 membered heterocyclyl, optionally substituted 3-10 membered heterocyclyl, optionally substituted 5-8 membered heterocyclyl, optionally substituted 5-6 membered heterocyclyl, optionally substituted 5-membered heterocyclyl, or optionally substituted 6-membered heterocyclyl. In certain embodiments, at least one instance of Rl is optionally substituted heterocyclyl, e.g., optionally substituted 3-14 membered heterocyclyl, optionally substituted 3-10 membered heterocyclyl, optionally substituted 5-8 membered heterocyclyl, optionally substituted 5-6 membered heterocyclyl, optionally substituted 5-membered heterocyclyl, or optionally substituted 6-membered heterocyclyl.
[0096] In some embodiments, Rl is optionally substituted aryl. In some embodiments, Rl is optionally substituted phenyl. In some embodiments, Rl is phenyl. In some embodiments, Rl is substituted phenyl. In certain embodiments, at least one instance of Rl is optionally substituted aryl, e.g., optionally substituted phenyl.
[0097] In some embodiments, Rl is optionally substituted heteroaryl, e.g., optionally substituted 5-14 membered heteroaryl, optionally substituted 5-10 membered heteroaryl, optionally substituted 5-6 membered heteroaryl, optionally substituted 5 membered heteroaryl, or optionally substituted 6 membered heteroaryl. In certain embodiments, at least one instance of Rl is optionally substituted heteroaryl, e.g., optionally substituted 5-14 membered heteroaryl, optionally substituted 5-10 membered heteroaryl, optionally substituted 5-6 membered heteroaryl, optionally substituted 5 membered heteroaryl, or optionally substituted 6 membered heteroaryl.
[0098] In some embodiments, Rl is halogen. In some embodiments, Rl is ¨F.
In some embodiments, Rl is ¨Cl. In some embodiments, Rl is ¨Br. In some embodiments, Rl is ¨I.
[0099] In some embodiments, Rl is ¨OR, wherein RA1 is as defined above and described herein. In some embodiments, Rl is ¨N(RA1)2, wherein each RA1 is independently as defined above and described herein. In some embodiments, Rl is ¨SRA1, wherein RA1 is as defined above and described herein.
[0100] In some embodiments, an Rl alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl group may be substituted. In some embodiments, an Rl alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl group may be substituted with an optionally substituted amino group. In some embodiments, an Rl alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl group may be substituted with an optionally substituted hydroxyl group. In some embodiments, an Rl alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl group may be substituted with an optionally substituted thiol group. In any of the above embodiments, an Rl alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl group may be substituted, for example, with an optionally substituted amino group (e.g., ¨NR6R7), an optionally substituted hydroxyl group (e.g., ¨0R6), an optionally substituted thiol group (e.g., ¨SR6), or with a group of formula (i), (ii), or (iii), wherein each instance of R6 and R7 is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, and a sulfur protecting group when attached to a sulfur atom, or a group of formula (i), (ii), or (iii).
[0101] In some embodiments, Rl is an optionally substituted natural amino acid side chain. In some embodiments, Rl is a natural amino acid side chain. In some embodiments, Rl is an optionally substituted unnatural amino acid side chain. In some embodiments, Rl is an unnatural amino acid side chain.
[0102] In certain embodiments, each instance of Rl is the same. In certain embodiments, at least one Rl group is different. In certain embodiments, each Rl group is different.
[0103] In certain embodiments, Rl is an alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl group substituted with an amino group of the formula ¨NR6R7.
[0104] In certain embodiments, Rl is a group of formula (iv):

r µ
R7 (iv) wherein:
L is an optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, or optionally substituted heteroarylene, or combination thereof; and each of R6 and R7 is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group, or a group of formula (i), (ii) or (iii):
R' R' XRL
RL --yRP
RL
_1 _I
(i) (ii) (iii) wherein each of R', Y, RP, RI- and X is independently as defined above and described herein.
[0105] In some embodiments, at least one instance of Rl is an alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl group substituted with an amino group of the formula ¨NR6R7. In some embodiments, at least one instance of Rl is a group of formula (iv).
In some embodiments, at least one instance of Rl is a group of formula (iv), wherein at least one instance of R6 and R7 is a group of the formula (i), (ii) or (iii). In some embodiments, at least one instance of Rl is a group of formula (iv), wherein each instance of R6 and R7 is a group of the formula (i), (ii) or (iii). In some embodiments, at least one instance of Rl is a group of formula (iv), wherein each instance of R6 and R7 is a group of the formula (i). In some embodiments, at least one instance of Rl is a group of formula (iv), wherein each instance of R6 and R7 is a group of the formula (ii). In some embodiments, at least one instance of Rl is a group of formula (iv), wherein each instance of R6 and R7 is a group of the formula (iii).
[0106] In some embodiments, each instance of Rl is a group of formula (iv). In some embodiments, each instance of Rl is a group of formula (iv), wherein each instance of R6 and R7 is a group of the formula (i), (ii) or (iii). In some embodiments, each instance of Rl is a group of formula (iv), wherein each instance of R6 and R7 is a group of the formula (i), (ii) or (iii). In some embodiments, each instance of Rl is a group of formula (iv), wherein each instance of R6 and R7 is a group of the formula (i). In some embodiments, each instance of Rl is a group of formula (iv), wherein each instance of R6 and R7 is a group of the formula (ii). In some embodiments, each instance of Rl is a group of formula (iv), wherein each instance of R6 and R7 is a group of the formula (iii).
[0107] In certain embodiments, at least two instances of Rl is a group of formula (iv). In certain embodiments, at least three instances of Rl is a group of formula (iv). In certain embodiments, at least four instances of Rl is a group of formula (iv). In certain embodiments, at least five instances of Rl is a group of formula (iv). In certain embodiments, at least six instances of Rl is a group of formula (iv). In certain embodiments, at least seven instances of Rl is a group of formula (iv). In certain embodiments, at least eight instances of Rl is a group of formula (iv). In certain embodiments, at least nine instances of Rl is a group of formula (iv). In certain embodiments, each instance of Rl is a group of formula (iv).
[0108] In certain embodiments, L is an optionally substituted alkylene;
e.g., optionally substituted Ci_50alkylene, optionally substituted Ci_40alkylene, optionally substituted Ci_ 30alkylene, optionally substituted Ci_20alkylene, optionally substituted C4_20alkylene, optionally substituted C6_20alkylene, optionally substituted C8_20alkylene, optionally substituted C10_ 20alkylene, optionally substituted Ci_6alkylene, optionally substituted C2_6alkylene, optionally substituted C3_6alkylene, optionally substituted C4_6alkylene, optionally substituted C4_5alkylene, or optionally substituted C3_4alkylene. In some embodiments, L is optionally substituted C 1 alkylene. In some embodiments, L is optionally substituted C2 alkylene. In some embodiments, L is optionally substituted C3 alkylene. In some embodiments, L is optionally substituted C4 alkylene. In some embodiments, L is optionally substituted C5 alkylene. In some embodiments, L is optionally substituted C6 alkylene. In some embodiments, L is optionally substituted C7 alkylene. In some embodiments, L is optionally substituted C8 alkylene. In some embodiments, L is optionally substituted C9 alkylene. In some embodiments, L is optionally substituted Cm alkylene. In some embodiments, L is -CH2-. In some embodiments, L is -(CH2)2-.
In some embodiments, L is -(CH2)3-. In some embodiments, L is -(CH2)4-. In some embodiments, L is -(CH2)5-. In some embodiments, L is -(CH2)6-. In some embodiments, L is -(CH2)7-. In some embodiments, L is -(CH2)8-. In some embodiments, L is -(CH2)9-. In some embodiments, L is -(CH2)10-.
[0109] In certain embodiments, L is an optionally substituted alkenylene, e.g., optionally substituted C2_50alkenylene, optionally substituted C2_40alkenylene, optionally substituted C2_ 30alkenylene, optionally substituted C2_20alkenylene, optionally substituted C4_20alkenylene, optionally substituted C6_20alkenylene, optionally substituted C8_20alkenylene, optionally substituted Cio-2oalkenylene, optionally substituted C2_6alkenylene, optionally substituted C3_ 6alkenylene, optionally substituted C4_6alkenylene, optionally substituted C4_5alkenylene, or optionally substituted C3_4alkenylene.
[0110] In certain embodiments, L is an optionally substituted alkynylene, e.g., optionally substituted C2_50alkynylene, optionally substituted C2_40alkynylene, optionally substituted C2_ 30alkynylene, optionally substituted C2_20alkynylene, optionally substituted C4_20alkynylene, optionally substituted C6_20alkynylene, optionally substituted C8_20alkynylene, optionally substituted Ci0_20alkynylene, optionally substituted C2_6alkynylene, optionally substituted C3_ 6alkynylene, optionally substituted C4_6alkynylene, optionally substituted C4_5alkynylene, or optionally substituted C3_4alkynylene.
[0111] In certain embodiments, L is an optionally substituted heteroalkylene; e.g., optionally substituted heteroCi_50alkylene, optionally substituted heteroCi_40alkylene, optionally substituted heteroCi_30alkylene, optionally substituted heteroCi_20alkylene, optionally substituted heteroC4_20alkylene, optionally substituted heteroC6_20alkylene, optionally substituted heteroC8_ 20alkylene, optionally substituted heteroCi0_20alkylene, optionally substituted heteroCi_6alkylene, optionally substituted heteroC2_6alkylene, optionally substituted heteroC3_6alkylene, optionally substituted heteroC4_6alkylene, optionally substituted heteroC4_5alkylene, or optionally substituted heteroC3_4alkylene. In some embodiments, L is optionally substituted heteroC2alkylene. In some embodiments, L is optionally substituted heteroC3alkylene. In some embodiments, L is optionally substituted heteroC4alkylene. In some embodiments, L is optionally substituted heteroC5alkylene. In some embodiments, L is optionally substituted heteroC6alkylene. In some embodiments, L is optionally substituted heteroC7alkylene. In some embodiments, L is optionally substituted heteroC8alkylene. In some embodiments, L is optionally substituted heteroC9alkylene. In some embodiments, L is optionally substituted heteroC 10 alkylene.
[0112] In certain embodiments, L is an optionally substituted heteroalkenylene, e.g., optionally substituted heteroC2_50alkenylene, optionally substituted heteroC2_40alkenylene, optionally substituted heteroC2_30alkenylene, optionally substituted heteroC2_20alkenylene, optionally substituted heteroC4_20alkenylene, optionally substituted heteroC6_20alkenylene, optionally substituted heteroC8_20alkenylene, optionally substituted heteroCio_20alkenylene, optionally substituted heteroC2_6alkenylene, optionally substituted heteroC3_6alkenylene, optionally substituted heteroC4_6alkenylene, optionally substituted heteroC4_5alkenylene, or optionally substituted heteroC3_4alkenylene.
[0113] In certain embodiments, L is an optionally substituted heteroalkynylene, e.g., optionally substituted heteroC2_50alkynylene, optionally substituted heteroC2_40alkynylene, optionally substituted heteroC2_30alkynylene, optionally substituted heteroC2_20alkynylene, optionally substituted heteroC4_20alkynylene, optionally substituted heteroC6_20alkynylene, optionally substituted heteroC8_20alkynylene, optionally substituted heteroCio_20alkynylene, optionally substituted heteroC2_6alkynylene, optionally substituted heteroC3_6alkynylene, optionally substituted heteroC4_6alkynylene, optionally substituted heteroC4_5alkynylene, or optionally substituted heteroC3_4alkynylene.
[0114] In certain embodiments, L is an optionally substituted carbocyclylene, e.g., optionally substituted C340carbocyclylene, optionally substituted C5_8carbocyclylene, optionally substituted C5_6carbocyclylene, optionally substituted C5carbocyclylene, or optionally substituted C6carbocyclylene.
[0115] In certain embodiments, L is an optionally substituted heterocyclylene, e.g., optionally substituted 3-14 membered heterocyclylene, optionally substituted 3-10 membered heterocyclylene, optionally substituted 5-8 membered heterocyclylene, optionally substituted 5-6 membered heterocyclylene, optionally substituted 5-membered heterocyclylene, or optionally substituted 6-membered heterocyclylene.
[0116] In certain embodiments, L is an optionally substituted arylene, e.g., optionally substituted phenylene. In some embodiments, L is optionally substituted phenylene. In some embodiments, L is substituted phenylene. In some embodiments, L is unsubstituted phenylene.
[0117] In certain embodiments, L is an optionally substituted heteroarylene, e.g., optionally substituted 5-14 membered heteroarylene, optionally substituted 5-10 membered heteroarylene, optionally substituted 5-6 membered heteroarylene, optionally substituted 5-membered heteroarylene, or optionally substituted 6-membered heteroarylene.
[0118] In certain embodiments, wherein L is an optionally substituted alkylene group, the ,seH,N,R7 group of formula (iv) is a group of the formula q , wherein q is an integer between 1 and 50, inclusive, and each of R6 and R7 is independently as defined above and described herein.
[0119] In certain embodiments, q is an integer between 1 and 40, inclusive. In certain embodiments, q is an integer between 1 and 30, inclusive. In certain embodiments, q is an integer between 1 and 20, inclusive. In certain embodiments, q is an integer between 1 and 10, inclusive. In certain embodiments, q is an integer between 4 and 20, inclusive. In certain embodiments, q is an integer between 6 and 20, inclusive. In certain embodiments, q is an integer between 2 and 10, inclusive. In certain embodiments, q is an integer between 2 and 9, inclusive. In certain embodiments, q is an integer between 2 and 8, inclusive.
In certain embodiments, q is an integer between 2 and 7, inclusive. In certain embodiments, q is an integer between 2 and 6, inclusive. In certain embodiments, q is an integer between 2 and 5, inclusive.
In certain embodiments, q is an integer between 2 and 4, inclusive. In certain embodiments, q is an integer between 3 and 10, inclusive. In certain embodiments, q is an integer between 3 and 8, inclusive. In certain embodiments, q is an integer between 3 and 7, inclusive.
In certain embodiments, q is an integer between 3 and 6, inclusive. In certain embodiments, q is an integer between 3 and 5, inclusive. In certain embodiments, q is 3 or 4. In certain embodiments, q is an integer between 3 and 9, inclusive. In certain embodiments, q is an integer between 8 and 20, inclusive. In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. In certain embodiments, q is 4. In certain embodiments, q is 5. In certain embodiments, q is 6. In certain embodiments, q is 7. In certain embodiments, q is 8. In certain embodiments, q is 9. In certain embodiments, q is 10.
[0120] As generally defined above, each R6 is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group, or a group of formula (i), (ii) or (iii).
[0121] In some embodiments, R6 is hydrogen.
[0122] In some embodiments, R6 is optionally substituted alkyl. In some embodiments, R6 is optionally substituted C2_50 alkyl. In some embodiments, R6 is optionally substituted C2_40 alkyl. In some embodiments, R6 is optionally substituted C2_30 alkyl. In some embodiments, R6 is optionally substituted C2_20 alkyl. In some embodiments, R6 is optionally substituted C2_19 alkyl. In some embodiments, R6 is optionally substituted C2_18 alkyl. In some embodiments, R6 is optionally substituted C2_17 alkyl. In some embodiments, R6 is optionally substituted C2_16 alkyl. In some embodiments, R6 is optionally substituted C2_15 alkyl. In some embodiments, R6 is optionally substituted C2_14 alkyl. In some embodiments, R6 is optionally substituted C2_13 alkyl. In some embodiments, R6 is optionally substituted C2_12 alkyl. In some embodiments, R6 is optionally substituted C2_11 alkyl. In some embodiments, R6 is optionally substituted C2_10 alkyl. In some embodiments, R6 is optionally substituted C2_9 alkyl. In some embodiments, R6 is optionally substituted C2_8 alkyl. In some embodiments, R6 is optionally substituted C2_7 alkyl.
In some embodiments, R6 is optionally substituted C2_6 alkyl.
[0123] In some embodiments, R6 is optionally substituted C4_50 alkyl. In some embodiments, R6 is optionally substituted C4_40 alkyl. In some embodiments, R6 is optionally substituted C4_30 alkyl. In some embodiments, R6 is optionally substituted C4_20 alkyl. In some embodiments, R6 is optionally substituted C4_19 alkyl. In some embodiments, R6 is optionally substituted C4_18 alkyl. In some embodiments, R6 is optionally substituted C4_17 alkyl. In some embodiments, R6 is optionally substituted C4_16 alkyl. In some embodiments, R6 is optionally substituted C4_15 alkyl. In some embodiments, R6 is optionally substituted C4_14 alkyl. In some embodiments, R6 is optionally substituted C4_13 alkyl. In some embodiments, R6 is optionally substituted C4_12 alkyl. In some embodiments, R6 is optionally substituted C4_11 alkyl. In some embodiments, R6 is optionally substituted C4_10 alkyl. In some embodiments, R6 is optionally substituted C4_9 alkyl. In some embodiments, R6 is optionally substituted C4_8 alkyl. In some embodiments, R6 is optionally substituted C4_7 alkyl. In some embodiments, R6 is optionally substituted C4_6 alkyl.
[0124] In some embodiments, R6 is optionally substituted C6_50 alkyl. In some embodiments, R6 is optionally substituted C6_40 alkyl. In some embodiments, R6 is optionally substituted C6_30 alkyl. In some embodiments, R6 is optionally substituted C6_20 alkyl. In some embodiments, R6 is optionally substituted C6_19 alkyl. In some embodiments, R6 is optionally substituted C6_18 alkyl. In some embodiments, R6 is optionally substituted C6_17 alkyl. In some embodiments, R6 is optionally substituted C6_16 alkyl. In some embodiments, R6 is optionally substituted C6_15 alkyl. In some embodiments, R6 is optionally substituted C6_14 alkyl. In some embodiments, R6 is optionally substituted C6_13 alkyl. In some embodiments, R6 is optionally substituted C6_12 alkyl. In some embodiments, R6 is optionally substituted C6_11 alkyl. In some embodiments, R6 is optionally substituted C6_10 alkyl. In some embodiments, R6 is optionally substituted C6_9 alkyl. In some embodiments, R6 is optionally substituted C6_8 alkyl. In some embodiments, R6 is optionally substituted C6_7 alkyl.
[0125] In some embodiments, R6 is optionally substituted C8_50 alkyl. In some embodiments, R6 is optionally substituted C8_40 alkyl. In some embodiments, R6 is optionally substituted C8_30 alkyl. In some embodiments, R6 is optionally substituted C8_20 alkyl. In some embodiments, R6 is optionally substituted C8_19 alkyl. In some embodiments, R6 is optionally substituted C8_18 alkyl. In some embodiments, R6 is optionally substituted C8_17 alkyl. In some embodiments, R6 is optionally substituted C8_16 alkyl. In some embodiments, R6 is optionally substituted C8_15 alkyl. In some embodiments, R6 is optionally substituted C8_14 alkyl. In some embodiments, R6 is optionally substituted C8_13 alkyl. In some embodiments, R6 is optionally substituted C8_12 alkyl. In some embodiments, R6 is optionally substituted C8_11 alkyl. In some embodiments, R6 is optionally substituted C8_10 alkyl. In some embodiments, R6 is optionally substituted C8_9 alkyl.
[0126] In some embodiments, R6 is optionally substituted C9_50 alkyl. In some embodiments, R6 is optionally substituted C9_40 alkyl. In some embodiments, R6 is optionally substituted C9_30 alkyl. In some embodiments, R6 is optionally substituted C9_20 alkyl. In some embodiments, R6 is optionally substituted C9_19 alkyl. In some embodiments, R6 is optionally substituted C9_18 alkyl. In some embodiments, R6 is optionally substituted C9_17 alkyl. In some embodiments, R6 is optionally substituted C9_16 alkyl. In some embodiments, R6 is optionally substituted C9_15 alkyl. In some embodiments, R6 is optionally substituted C9_14 alkyl. In some embodiments, R6 is optionally substituted C9_13 alkyl. In some embodiments, R6 is optionally substituted C9_12 alkyl. In some embodiments, R6 is optionally substituted C9_11 alkyl. In some embodiments, R6 is optionally substituted C9_10 alkyl.
[0127] In some embodiments, R6 is optionally substituted C100 alkyl. In some embodiments, R6 is optionally substituted Cio-4o alkyl. In some embodiments, R6 is optionally substituted Cio_30 alkyl. In some embodiments, R6 is optionally substituted C1o_20 alkyl. In some embodiments, R6 is optionally substituted C10-19 alkyl. In some embodiments, R6 is optionally substituted Cio_ig alkyl. In some embodiments, R6 is optionally substituted C1o_17 alkyl. In some embodiments, R6 is optionally substituted Cio-16 alkyl. In some embodiments, R6 is optionally substituted C10_15 alkyl. In some embodiments, R6 is optionally substituted C1o_14 alkyl. In some embodiments, R6 is optionally substituted C10-13 alkyl. In some embodiments, R6 is optionally substituted C1o_12 alkyl. In some embodiments, R6 is optionally substituted C10_11 alkyl.
[0128] In some embodiments, R6 is optionally substituted C11-50 alkyl. In some embodiments, R6 is optionally substituted C 1 1-4o alkyl. In some embodiments, R6 is optionally substituted Ci1_30 alkyl. In some embodiments, R6 is optionally substituted C11_20 alkyl. In some embodiments, R6 is optionally substituted C11-19 alkyl. In some embodiments, R6 is optionally substituted C1118 alkyl. In some embodiments, R6 is optionally substituted Ci 1_17 alkyl. In some embodiments, R6 is optionally substituted C 1 146 alkyl. In some embodiments, R6 is optionally substituted Cii_i5 alkyl. In some embodiments, R6 is optionally substituted C11_14 alkyl. In some embodiments, R6 is optionally substituted C11-13 alkyl. In some embodiments, R6 is optionally substituted C11-12 alkyl.
[0129] In some embodiments, R6 is optionally substituted C12_50 alkyl. In some embodiments, R6 is optionally substituted C12-40 alkyl. In some embodiments, R6 is optionally substituted Ci2_30 alkyl. In some embodiments, R6 is optionally substituted C12_20 alkyl. In some embodiments, R6 is optionally substituted C12-19 alkyl. In some embodiments, R6 is optionally substituted C12_18 alkyl. In some embodiments, R6 is optionally substituted C12_17 alkyl. In some embodiments, R6 is optionally substituted C12-16 alkyl. In some embodiments, R6 is optionally substituted C12_15 alkyl. In some embodiments, R6 is optionally substituted C12_14 alkyl. In some embodiments, R6 is optionally substituted C12-13 alkyl.
[0130] In some embodiments, R6 is optionally substituted C6 alkyl. In some embodiments, R6 is optionally substituted C7 alkyl. In some embodiments, R6 is optionally substituted C8 alkyl. In some embodiments, R6 is optionally substituted C9 alkyl. In some embodiments, R6 is optionally substituted C10 alkyl. In some embodiments, R6 is optionally substituted C 1 1 alkyl. In some embodiments, R6 is optionally substituted C12 alkyl. In some embodiments, R6 is optionally substituted C13 alkyl. In some embodiments, R6 is optionally substituted C14 alkyl. In some embodiments, R6 is optionally substituted C15 alkyl. In some embodiments, R6 is optionally substituted C16 alkyl. In some embodiments, R6 is optionally substituted C17 alkyl. In some embodiments, R6 is optionally substituted C18 alkyl. In some embodiments, R6 is optionally substituted C19 alkyl. In some embodiments, R6 is optionally substituted C20 alkyl.
[0131] In some embodiments, for example, in any of the above embodiments, R6 is a substituted alkyl group. In some embodiments, R6 is an unsubstituted alkyl group. In some embodiments, R6 is an optionally substituted straight-chain alkyl group. In some embodiments, R6 is a substituted straight-chain alkyl group. In some embodiments, R6 is an unsubstituted straight-chain alkyl group. In some embodiments, R6 is an optionally substituted branched alkyl group. In some embodiments, R6 is a substituted branched alkyl group. In some embodiments, R6 is an unsubstituted branched alkyl group.
[0132] In some embodiments, R6 is optionally substituted alkenyl. In some embodiments, R6 is optionally substituted C2_50 alkenyl. In some embodiments, R6 is optionally substituted C2_40 alkenyl. In some embodiments, R6 is optionally substituted C2_30 alkenyl. In some embodiments, R6 is optionally substituted C2_20 alkenyl. In some embodiments, R6 is optionally substituted C2_19 alkenyl. In some embodiments, R6 is optionally substituted C2-18 alkenyl. In some embodiments, R6 is optionally substituted C2_17 alkenyl. In some embodiments, iCupuopdo sT 91 `sluouupoquio mos ui 11Cuo3HE zI-9D pouupsqns iCipuopdo ST 91 `sumuupoquio mos ui liCuo3HE EI-9D pouupsqns iCupuopdo ST ?I `sluouupoquio mos ui liCuo3HE
tI
-9D pouupsqns iCipuopdo ST 91 `sumuupoquio mos ui 1iCuo3HE cI-9D pouupsqns iCipuopdo sT 91 `sluouupoquio mos ui 1iCuo3HE 9I-9D popupsqns iCupuopdo ST 91 `sumuupoquio mos ui 11Cuo3HE
LI-9D pouupscins iCipuopdo ST ?I `sumuupoquio mos ui 1Jcuo3HE 8 1-9D
pouupscins iCipuopdo ST 911 `sumuupoquio mos ui 1Jcuo3HE 61-9D pouupscins iCipuopdo ST 911 `sluouupoquio mos uI .1,Cuo3IIP oz-9D pouupscins iCipuopdo ST 91 `sumuupoquio mos ui .pcuo3up E-9D pouupscins iCupuopdo ST 91 `sluouupoquio mos ui 1iCuo3up 9t-9D pouupsqns iCipuopdo ST 91 `sumuupoquio mos ui .pcuo3up os-9D pouupscins iCipuopdo ST 911 `sumuupoquio mos ui Irciol .pcuo3up 9-tp popupsqns iCupuopdo ST 91 `sumuupoquio mos ui 1iCuo3up L.-fp pouupscins iCipuopdo ST 91 `sumuupoquio aims ui .pcuo3up 8-17D
pouupscins iCipuopdo ST ?I `sumuupoquio mos ui liCu03up 6-173 pouupsqns iCipuopdo ST ?I
`sluouupoquio mos uI *IiCu03IIP in-fp pouupscins iCipuopdo ST 91 `sumuupoquio mos ui .pcu03up 11-17D pouupscins iCupuopdo ST 91 `sluouupoquio mos ui 1iCuo3up zI-17D pouupsqns iCipuopdo ST
91 `sumuupoquio mos ui liCu03up EI-tD pouupsqns iCupuopdo ST ?I `sluouupoquio mos ui liCu03up tI
-17D pouupsqns iCipuopdo ST 91 `sumuupoquio mos ui 1iCuo3up cI-to pouupsqns iCipuopdo sT 91 `sluouupoquio mos ui liCu03up 9I-tD popupsqns iCupuopdo ST 91 `sumuupoquio mos ui 1iCuo3up LTD pouupscins iCipuopdo ST ?I `sumuupoquio mos ui 1Jcuo3up ST-tp pouupscins iCipuopdo ST 911 `sumuupoquio mos ui .pcu03up 6i-tD pouupscins iCipuopdo ST 911 `sluouupoquio mos uI *IiCu03IIP oz-tp pouupscins iCipuopdo ST 91 `sumuupoquio mos ui .pcu03up 0E-tD pouupscins iCupuopdo ST 91 `sluouupoquio mos ui 1iCuo3up 017-to pouupsqns iCipuopdo ST
91 `sumuupoquio mos ui .pcu03up os-tD pouupscins iCipuopdo ST 911 `sumuupoquio mos ui 1I0]
1iCuo3up 9-zD pouupsqns iCipuopdo sT 91 `sluouupoquio mos ui 11Cu03up L-ZD popupsqns iCupuopdo ST 91 `sumuupoquio mos ui 1iCuo3up 8-ZD pouupsqns iCipuopdo ST 91 `sumuupoquio mos ui 1iCuo3up 6-zD pouupsqns iCipuopdo ST 911 `sumuupoquio mos ui 1Jcuo3up (ThzD pouupscins iCipuopdo ST 911 `sluouupoquio mos uI *IiCuo3IIP II-zD pouupsqns iCipuopdo ST 91 `sumuupoquio mos ui 11Cu03up zI-zD pouupsqns iCupuopdo ST 91 `sluouupoquio mos ui 1iCuo3up EI-zD pouupsqns iCipuopdo ST 91 `sumuupoquio mos ui 1iCuo3up 171-zD pouupsqns iCupuopdo ST ?I `sluouupoquio mos ui 1iCuo3up cI
-zp pouupsqns iCipuopdo ST 91 `sumuupoquio mos ui 1iCuo3up 9I-zD pouupsqns iCipuopdo sT 91 6LI90/tIOZSI1IIDd L9tI90/SIOZ OM

Or Li-oiD palmpscins iCipuopdo ST 911 `siuoulTpoqulo alms ui .pcuo3HE si-oiD
palmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui 1icuo3HE 6I-0I D palmpscins iCipuopdo ST 911 `sluoulTpoqulo mos uI .1,Cuo3IIP oz-oID pompscins iCupuopdo ST 911 `siuoulTpoqulo aims ui .pcu03up cis-imp polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui 1icuo3up of-oID polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui licu03up os-oID palmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui UN]
licuolip 01-6D polmpscins iCipuopdo ST 91 `siuoulTpoqulo mos ui 1icuo3up I1-6D polmpscins ictpuopdo ST 911 `siuoulTpoqulo mos ui 1icuo3up zI-6D polmpscins iCipuopdo ST
911 `siuoulTpoqulo alms ui liCu03up EI-6D polmpscins iCupuopdo ST 911 `siuoulTpoqulo alms ui liCu03up tI
-6D polmRscins icipuopdo ST 911 `siuoulTpoqulo alms ui .pcu03up cI-6D
pompscins icipuopdo sT 911 `siuoulTpoqulo alms ui 1Jcuo3up 91-6D polmpscins iCupuopdo ST 91 `siuoulTpoqulo alms ui 11Cuo3up LI-6D polmpscins iCipuopdo ST ?I `siuoulTpoqulo alms ui .pcu03up 8 '6D
polmpscins iCipuopdo ST 911 `siuoulTpoqulo alms ui licu03up 6I-6D polmpscins iCipuopdo ST 911 `siuoulTpoqulo alms uI *IiCu03IIP oz-6D polmpscins iCipuopdo ST 91 `siuoulTpoqulo alms ui licu03up 9E-6D polmpscins ictpuopdo ST 911 `siuoulTpoqulo alms ui 1icuo3up (0-6D polmpscins iCipuopdo ST
911 `siuoulTpoqulo alms ui 1Jcuo3up os-6D polmpscins iCipuopdo ST 911 `siuoulTpoqulo alms ui 19E10]
licuolip 6-8D polmpscins iCipuopdo ST 911 `siuoulTpoqulo alms uI *IiCuo3IIP o-r-sD polmpscins iCipuopdo ST 91 `siuoulTpoqulo alms ui 11cu03up II-8D polmpscins iCupuopdo ST 91 `siuoulTpoqulo alms ui 1iCuo3up zI-8D polmpsqns iCipuopdo ST
91 `siuoulTpoqulo alms ui 1iCuo3up EI-8D polmpsqns iCupuopdo ST ?I `siuoulTpoqulo alms ui 1iCuo3up tI
-8D polmpsqns iCipuopdo ST 91 `siuoulTpoqulo alms ui 1iCuo3up cI-8D pompsqns iCipuopdo sT 91 `siuoulTpoqulo alms ui 1iCuo3up 91-8D polmpsqns iCupuopdo ST 91 `siuoulTpoqulo alms ui 1iCuo3up LI-sD polmpscins iCipuopdo ST ?I `siuoulTpoqulo alms ui 1Jcuo3up 81-8D
polmpscins iCipuopdo ST 911 `siuoulTpoqulo alms ui 1Jcuo3up 61-8D polmpscins iCipuopdo ST 911 `siuoulTpoqulo alms uI *IiCuo3IIP oz-sD polmpscins iCipuopdo ST 91 `siuoulTpoqulo alms ui 11cu03up (/E-8D polmpscins iCupuopdo ST 91 `siuoulTpoqulo alms ui 1iCuo3up 017-8D polmpsqns iCipuopdo ST
91 `siuoulTpoqulo alms ui 1Jcuo3up 0s-8D polmpscins iCipuopdo ST 911 `siuoulTpoqulo alms ui ISM]
.pcuo3up L-oD polmpscins iCipuopdo ST 91 `siuoulTpoqulo alms ui 1icuo3up 8-9D
polmpscins iCipuopdo ST ?i `siuoulTpoqulo alms ui 1iCuo3up 6-9D polmpsqns iCipuopdo ST ?I
`siuoulTpoqulo alms uI *I1Cu03IIP moD polmpscins iCipuopdo ST 91 `siuoulTpoqulo alms ui 11cu03up IT-9D polmpscins 6LI90/tIOZSI1IIDd L9tI90/SIOZ OM

It JCII1II0pCIO SI ?I `siuoulTpoqulo mos ui =pCuo3HE 91D polmpsqns iCipuopdo ST
91 `siuoulTpoqulo mos ui =pCuo3HE cID polmpsqns iCipuopdo ST ?I `siuoulTpoqulo aims ui =pCu03HE
HD polmpsqns iCipuopdo ST ?I `siuoulTpoqulo mos ui 1JCuo3HE LID polmpsqns iCipuopdo ST 91 `siuoulTpoqulo aims ui =pCu03HE zip polmpsqns iCipuopdo ST ?i `siuoulTpoqulo aims ui =pCu03HE
iip polmpsqns icipuopdo ST 911 `siuoulTpoqulo mos ui 1Jcuo3HE ID polmpscins iCipuopdo ST
911 `siuoulTpoqulo aims ui =pCu03HE 6D pompsqns iCipuopdo ST 9i `siuoulTpoqulo mos ui =pCu03HE
SD polmpsqns iCipuopdo ST 91 `siuoulTpoqulo mos ui 1JCu03HE LD polmpsqns iCipuopdo ST 91 `siuoulTpoqulo mos ui =pCu03HE 9D polmpsqns iCipuopdo ST ?I `siuoulTpoqulo mos ui [ono]
lictio3HE EI-zID pompsqns iCupuopdo ST ?I `siuoulTpoqulo mos ui 1JCu03HE p polmpsqns iCipuopdo ST 91 `siuoulTpoqulo mos ui 1JCu03HE ci-zID polmpsqns iCipuopdo ST
91 `siuoulTpoqulo mos ui =pCu03HE 9I-zID polmpsqns iCupuopdo ST 91 `siuoulTpoqulo mos ui =pCu03HE
Li-ziD polmpscins iCipuopdo ST 911 `siuoulTpoqulo MOS III 1ic11.03HE SI-ZID
polmpscins 1CIIPII0pd0 ST II `siuoulTpoqulo mos ui Itc1103HE 6T-ZID polmpscins 1CIIPII0pd0 ST II
`siuoulTpoqulo mos UI .I1C11 )IF OZ-ZID pompscins iCupuopdo ST 911 `siuoulTpoqulo mos ui 1Jcuo3up os-ziD polmpscins icipuopdo ST 911 `siuoulTpoqulo mos ui 1Jcuo3up ot-zID palmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui 1Jcuo3up os-ziD palmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui 16E10]
1JCuo3up zI-Iip polmpsqns iCipuopdo ST ?I `siuoulTpoqulo mos UI *IiCuo3IIP EI-Iip pompsqns iCupuopdo ST ?I `siuoulTpoqulo mos ui 1JCuo3up tI-Iip polmpsqns iCipuopdo ST 91 `siuoulTpoqulo mos ui 1JCuo3up cI-IID polmpsqns iCipuopdo ST
91 `siuoulTpoqulo mos ui 1JCuo3up 91-11D polmpsqns iCupuopdo ST 91 `siuoulTpoqulo mos ui 1JCuo3up Li-i ID polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui 1Jcuo3up ID polmpscins JCIIPII0pd0 ST II `siuoulTpoqulo mos ui 1Jcuo3up 61-11D palmpscins JCIIPII0pd0 ST II
`siuoulTpoqulo mos UI .I1C11 )IF T TD pompscins iCupuopdo ST 911 `siuoulTpoqulo mos ui 1Jcuo3up 0-I ID polmpscins icipuopdo ST 911 `siuoulTpoqulo mos ui 1Jcuo3up ot-TID palmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui 1JCuo3up 0c-11D polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui 1810]
1Jcuo3up 1 i-oID palmpscins iCipuopdo ST 9-N `siuoulTpoqulo mos ui 1Jcuo3up zi-oi D palmpscins iCipuopdo ST 911 `siuoulTpoqulo mos UI *I1Cu03IIP si-oiD pompscins iCupuopdo ST 911 `siuoulTpoqulo mos ui 1Jcuo3up ti-oID polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui 1Jcuo3up si-oi D polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui =pcu03up 91-61D palmpscins iCupuopdo ST 911 `siuoulTpoqulo mos ui 1JCuo3up 6LI90/tIOZSI1IIDd L9tI90/SIOZ OM

substituted C17 alkenyl. In some embodiments, R6 is optionally substituted C18 alkenyl. In some embodiments, R6 is optionally substituted C19 alkenyl. In some embodiments, R6 is optionally substituted C20 alkenyl.
[0141] In some embodiments, for example, in any of the above embodiments, R6 is a substituted alkenyl group. In some embodiments, R6 is an unsubstituted alkenyl group. In some embodiments, R6 is an optionally substituted straight-chain alkenyl group. In some embodiments, R6 is a substituted straight-chain alkenyl group. In some embodiments, R6 is an unsubstituted straight-chain alkenyl group. In some embodiments, R6 is an optionally substituted branched alkenyl group. In some embodiments, R6 is a substituted branched alkenyl group. In some embodiments, R6 is an unsubstituted branched alkenyl group.
[0142] In some embodiments, R6 is optionally substituted alkynyl. In some embodiments, R6 is optionally substituted C2_50 alkynyl. In some embodiments, R6 is optionally substituted C2_40 alkynyl. In some embodiments, R6 is optionally substituted C2_30 alkynyl. In some embodiments, R6 is optionally substituted C2_20 alkynyl. In some embodiments, R6 is optionally substituted C2_19 alkynyl. In some embodiments, R6 is optionally substituted C2_18 alkynyl. In some embodiments, R6 is optionally substituted C2_17 alkynyl. In some embodiments, R6 is optionally substituted C2_16 alkynyl. In some embodiments, R6 is optionally substituted C2_15 alkynyl. In some embodiments, R6 is optionally substituted C2_14 alkynyl. In some embodiments, R6 is optionally substituted C2_13 alkynyl. In some embodiments, R6 is optionally substituted C2_12 alkynyl. In some embodiments, R6 is optionally substituted C2_11 alkynyl. In some embodiments, R6 is optionally substituted C2_10 alkynyl. In some embodiments, R6 is optionally substituted C2_9 alkynyl. In some embodiments, R6 is optionally substituted C2_8 alkynyl. In some embodiments, R6 is optionally substituted C2_7 alkynyl. In some embodiments, R6 is optionally substituted C2_6 alkynyl.
[0143] In some embodiments, R6 is optionally substituted C4_50 alkynyl.
In some embodiments, R6 is optionally substituted C4_40 alkynyl. In some embodiments, R6 is optionally substituted C4_30 alkynyl. In some embodiments, R6 is optionally substituted C4_20 alkynyl. In some embodiments, R6 is optionally substituted C4_19 alkynyl. In some embodiments, R6 is optionally substituted C4_18 alkynyl. In some embodiments, R6 is optionally substituted C4_17 alkynyl. In some embodiments, R6 is optionally substituted C4_16 alkynyl. In some a ST 91 `siuoulTpoqulo aims ui 1iCuiC3HE z1-83 polmpsqns iCipuopdo ST ?i `sluoulTpoqulo mos uI *IiCuiC3IIP EI-83 polmpsqns iCupuopdo sT ?i `siuoulTpoqulo mos ui 1iCuiC3up I71-8D polmpsqns iCipuopdo sT 91 `siuoulTpoqulo mos ui liCu1C3up c1-8D polmpsqns iCipuopdo sT
91 `siuoulTpoqulo mos ui 1iCuiC3up 91-8D polmpsqns iCipuopdo sT ?I `siuoulTpoqulo mos ui 1iCuiC3up LI-sp polmpscins iCipuopdo sT 911 `sluoulTpoqulo mos ui 1iCuic3up 81-8D
polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui 1iCuic3up 6T-8D polmpscins iCipuopdo ST 911 `sluoulTpoqulo mos uI *IiCuiC3IIP oz-sp polmpscins iCtpuopdo ST 911 `siuoulTpoqulo aims ui liCu1c3up 0E-8D polmpscins icipuopdo ST 91 `siuoulTpoqulo mos ui 1iCuic3up ot-8D polmpscins iCipuopdo ST
911 `siuoulTpoqulo mos ui liCu1c3up os-8D polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui Istio]
.pcuic3up L-9D polmpscins iCipuopdo ST 911 `siuoulTpoqulo aims ui liCuic3up 8-9D
pompscins iCipuopdo sT 911 `siuoulTpoqulo mos ui 1iCuiC3up 6-9D polmpsqns iCipuopdo ST ?I `siuoulTpoqulo mos ui 11Cu1C3up oT-9D polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui liCuic3up H-9D
polmpscins iCipuopdo ST 91 `siuoulTpoqulo mos ui 1iCuiC3up z1-9D polmpsqns iCipuopdo ST ?I
`siuoulTpoqulo mos uI *IiCu1C3IIP 1-9D polmpsqns iCupuopdo ST ?I `siuoulTpoqulo mos ui liCu1C3up tI-9D polmpsqns iCipuopdo ST 91 `siuoulTpoqulo mos ui 1iCuiC3up cI-9D polmpsqns iCipuopdo ST
91 `siuoulTpoqulo mos ui liCu1C3up 91-9D polmpsqns iCipuopdo ST ?I `siuoulTpoqulo mos ui liCu1C3up LI-9D polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui 1iCuic3up 8 1-9D
polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui liCu1c3up 61-9D polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos uI *IiCuiC3IIP oz-9D polmpscins iCtpuopdo ST 911 `siuoulTpoqulo mos ui liCuic3up 0E-9D polmpscins icipuopdo ST 91 `siuoulTpoqulo mos ui liCu1c3up ot-9D polmpscins iCipuopdo ST
911 `siuoulTpoqulo mos ui 1iCuic3up os-9D polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui [trio]
1Jcuic3up 9-tp polmpsqns iCipuopdo ST ?I `siuoulTpoqulo mos ui liCu1C3up L.-fp polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui 1iCuic3up 8-tD
pompscins iCipuopdo sT 911 `siuoulTpoqulo mos ui liCu1C3up 610 polmpsqns iCipuopdo ST ?I `siuoulTpoqulo mos ui liCu1C3up oi-tp polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui liCuic3up "D
polmpscins iCipuopdo ST 91 `siuoulTpoqulo mos ui 1iCuiC3up D polmpsqns iCipuopdo ST ?I
`siuoulTpoqulo mos uI *IiCuiC3IIP EI-tD polmpsqns iCupuopdo ST ?I `siuoulTpoqulo mos ui liCuiC3up tI-tD polmpsqns iCipuopdo ST 91 `siuoulTpoqulo mos ui 1iCuiC3up cI-17D polmpsqns iCipuopdo ST
91 `siuoulTpoqulo 6LI90/tIOZSI1IIDd L9tI90/SIOZ OM

Ii, JCIIPUOTWO ST 91 `siuoulTpoqulo mos ui 1iCuiC3HE cI-11D polmpsqns iCipuopdo sT 91 `siuoulTpoqulo mos ui liCu1C3HE 9I-IID polmpsqns iCipuopdo sT ?i `siuoulTpoqulo mos ui liCu1C3HE
Li-iiD palmpscins iCtpuopdo sT 911 `siuoulTpoqulo mos ui licu1c3HE sTT ID
palmpscins iCipuopdo ST II `sluoulTpoqulo mos ui liclitc3HE 61-11D pop lmscins iCipuopdo ST II `sluoulTpoqulo mos . 9 . 9 UT .IICTIA3TIP ' 'DpalmRscins iCipuopdo ST 911 `siuoulTpoqulo mos ui .pcu1c3up oE-TID polmpscins icipuopdo ST 911 `siuoulTpoqulo mos ui 1Jcuic3up ot-iiD palmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui .pcu1c3up os-HD palmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui 18tI0]
1icuic3up " 'D polmpscins iCipuopdo ST II `sluoulTpoqulo mos ui .pcuic3up z T-oiD palmpscins iCipuopdo ST II
`sluoulTpoqulo mos . 9 . 9 UT .IICTIA3TIP EI-OID palmRscins iCipuopdo ST 911 `siuoulTpoqulo aims ui 1Jcuic3up ti-oiD polmpscins icipuopdo ST 911 `siuoulTpoqulo mos ui 1Jcuic3up sT-oID palmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui licu1c3up 91-oiD polmRscins iCipuopdo ST 911 `siuoulTpoqulo mos ui liCu1C3up Li-oiD polmpscins iCtpuopdo ST 911 `siuoulTpoqulo mos ui 1icuic3up sT-oID
polmpscins iCipuopdo ST II `sluoulTpoqulo mos ui .pcuic3up 61-oiD palmpscins iCipuopdo ST II
`sluoulTpoqulo mos . 9 . 9 UT .IICTI13TIP OZ-OID palmRscins iCipuopdo ST 911 `siuoulTpoqulo mos ui .pcu1c3up oE-oiD polmpscins icipuopdo ST 911 `siuoulTpoqulo mos ui 1Jcuic3up ot-oiD palmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui licu1c3up os-oID polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui [cm]
.pcuic3up oT-6D polmpscins iCipuopdo ST 911 `sluoulTpoqulo mos ui liCu1c3up H-6D
polmpscins iCipuopdo ST II `siuoulTpoqulo mos ui liCuic3up zI-6D polmpscins iCipuopdo ST II
`sluoulTpoqulo mos . 9 . 9 UI .IICTI13TIP E1-6D polmpscins iCtpuopdo ST 911 `siuoulTpoqulo mos ui liCu1c3up tI-6D polmpscins icipuopdo ST 91 `siuoulTpoqulo mos ui 1iCuic3up si-6D polmpscins iCipuopdo ST
911 `siuoulTpoqulo mos ui liCu1c3up 91-6D polmpscins iCipuopdo ST ?I `siuoulTpoqulo mos ui liCu1C3up LI-6D polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui liCu1c3up 81-6D
polmpscins iCipuopdo ST II `siuoulTpoqulo mos ui liCuic3up 61-6D polmpscins iCipuopdo ST II
`siuoulTpoqulo mos . 9 . 9 UI .IICTI13TIP OZ-6D polmpscins iCtpuopdo ST 911 `siuoulTpoqulo mos ui liCu1c3up 0E-6D polmpscins icipuopdo ST 91 `siuoulTpoqulo mos ui 1iCuic3up ot-6D polmpscins iCipuopdo ST
911 `siuoulTpoqulo mos ui liCu1c3up os-6D polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui 19tI0]
licu1c3up 6-8D polmpscins iCipuopdo ST ?I `siuoulTpoqulo mos ui 1iCuiC3up oi-sD polmpscins iCipuopdo ST 911 `siuoulTpoqulo mos ui 1iCuic3up IT-8D
polmpscins iCipuopdo 6LI90/tIOZSI1IIDd L9tI90/SIOZ OM

substituted Ci1_14 alkynyl. In some embodiments, R6 is optionally substituted C11_13 alkynyl. In some embodiments, R6 is optionally substituted C11-12 alkynyl.
[0149] In some embodiments, R6 is optionally substituted C12-50 alkynyl.
In some embodiments, R6 is optionally substituted Cu-4o alkynyl. In some embodiments, R6 is optionally substituted Ci2_30 alkynyl. In some embodiments, R6 is optionally substituted C12_20 alkynyl. In some embodiments, R6 is optionally substituted C12-19 alkynyl. In some embodiments, R6 is optionally substituted C12_18 alkynyl. In some embodiments, R6 is optionally substituted C12_17 alkynyl. In some embodiments, R6 is optionally substituted C12-16 alkynyl. In some embodiments, R6 is optionally substituted C12-15 alkynyl. In some embodiments, R6 is optionally substituted C1214 alkynyl. In some embodiments, R6 is optionally substituted C12_13 alkynyl.
[0150] In some embodiments, R6 is optionally substituted C6 alkynyl. In some embodiments, R6 is optionally substituted C7 alkynyl. In some embodiments, R6 is optionally substituted C8 alkynyl. In some embodiments, R6 is optionally substituted C9 alkynyl. In some embodiments, R6 is optionally substituted C10 alkynyl. In some embodiments, R6 is optionally substituted Cii alkynyl. In some embodiments, R6 is optionally substituted C12 alkynyl. In some embodiments, R6 is optionally substituted C13 alkynyl. In some embodiments, R6 is optionally substituted C14 alkynyl. In some embodiments, R6 is optionally substituted C15 alkynyl. In some embodiments, R6 is optionally substituted C16 alkynyl. In some embodiments, R6 is optionally substituted C17 alkynyl. In some embodiments, R6 is optionally substituted C18 alkynyl. In some embodiments, R6 is optionally substituted C19 alkynyl. In some embodiments, R6 is optionally substituted C20 alkynyl.
[0151] In some embodiments, for example, in any of the above embodiments, R6 is a substituted alkynyl group. In some embodiments, R6 is an unsubstituted alknyl group. In some embodiments, R6 is an optionally substituted straight-chain alkynyl group. In some embodiments, R6 is a substituted straight-chain alkynyl group. In some embodiments, R6 is an unsubstituted straight-chain alkynyl group. In some embodiments, R6 is an optionally substituted branched alkynyl group. In some embodiments, R6 is a substituted branched alkynyl group. In some embodiments, R6 is an unsubstituted branched alkynyl group.

[0152] In some embodiments, R6 is optionally substituted carbocyclyl. In some embodiments, R6 is optionally substituted heterocyclyl. In some embodiments, R6 is optionally substituted aryl. In some embodiments, R6 is optionally substituted heteroaryl. In some embodiments, R6 is a nitrogen protecting group.
[0153] In some embodiments, R6 is a group of formula (i). In some embodiments, R6 is a RL
_)--OH
group of formula (i-a). In some embodiments, R6 is a group of formula ¨1 (i-al). In some embodiments, R6 is a group of formula (i-b). In some embodiments, R6 is a group of formula (ii). In some embodiments, R6 is a group of formula (iii).
[0154] In some embodiments, R6 is substituted with one or more hydroxyl groups. In some embodiments, R6 is substituted with one hydroxyl group. In some embodiments, R6 is substituted with one 2-hydroxyl group (Cl is the carbon atom directly bonded to the nitrogen atom depicted in formula (iv)).
[0155] As generally defined above, each R7 is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group, or a group of formula (i), (ii) or (iii).
[0156] In some embodiments, R7 is hydrogen.
[0157] In some embodiments, R7 is optionally substituted alkyl. In some embodiments, R7 is optionally substituted C2_50 alkyl. In some embodiments, R7 is optionally substituted C2_40 alkyl. In some embodiments, R7 is optionally substituted C2_30 alkyl. In some embodiments, R7 is optionally substituted C2_20 alkyl. In some embodiments, R7 is optionally substituted C2_19 alkyl. In some embodiments, R7 is optionally substituted C2_18 alkyl. In some embodiments, R7 is optionally substituted C2_17 alkyl. In some embodiments, R7 is optionally substituted C2_16 alkyl. In some embodiments, R7 is optionally substituted C2_15 alkyl. In some embodiments, R7 is optionally substituted C2_14 alkyl. In some embodiments, R7 is optionally substituted C2_13 alkyl. In some embodiments, R7 is optionally substituted C2_12 alkyl. In some embodiments, R7 is optionally substituted C2_11 alkyl. In some embodiments, R7 is optionally substituted C2_10 alkyl. In some embodiments, R7 is optionally substituted C2_9 alkyl. In some embodiments, R7 is optionally substituted C2_8 alkyl. In some embodiments, R7 is optionally substituted C2_7 alkyl.
In some embodiments, R7 is optionally substituted C2_6 alkyl.
[0158] In some embodiments, R7 is optionally substituted C4_50 alkyl. In some embodiments, R7 is optionally substituted C4_40 alkyl. In some embodiments, R7 is optionally substituted C4_30 alkyl. In some embodiments, R7 is optionally substituted C4_20 alkyl. In some embodiments, R7 is optionally substituted C4_19 alkyl. In some embodiments, R7 is optionally substituted C4_18 alkyl. In some embodiments, R7 is optionally substituted C4_17 alkyl. In some embodiments, R7 is optionally substituted C4_16 alkyl. In some embodiments, R7 is optionally substituted C4_15 alkyl. In some embodiments, R7 is optionally substituted C4_14 alkyl. In some embodiments, R7 is optionally substituted C4_13 alkyl. In some embodiments, R7 is optionally substituted C4_12 alkyl. In some embodiments, R7 is optionally substituted C4_11 alkyl. In some embodiments, R7 is optionally substituted C4_10 alkyl. In some embodiments, R7 is optionally substituted C4_9 alkyl. In some embodiments, R7 is optionally substituted C4_8 alkyl. In some embodiments, R7 is optionally substituted C4_7 alkyl. In some embodiments, R7 is optionally substituted C4_6 alkyl.
[0159] In some embodiments, R7 is optionally substituted C6_50 alkyl. In some embodiments, R7 is optionally substituted C6_40 alkyl. In some embodiments, R7 is optionally substituted C6_30 alkyl. In some embodiments, R7 is optionally substituted C6_20 alkyl. In some embodiments, R7 is optionally substituted C6_19 alkyl. In some embodiments, R7 is optionally substituted C6_18 alkyl. In some embodiments, R7 is optionally substituted C6_17 alkyl. In some embodiments, R7 is optionally substituted C6_16 alkyl. In some embodiments, R7 is optionally substituted C6_15 alkyl. In some embodiments, R7 is optionally substituted C6_14 alkyl. In some embodiments, R7 is optionally substituted C6_13 alkyl. In some embodiments, R7 is optionally substituted C6_12 alkyl. In some embodiments, R7 is optionally substituted C6_11 alkyl. In some embodiments, R7 is optionally substituted C6_10 alkyl. In some embodiments, R7 is optionally substituted C6_9 alkyl. In some embodiments, R7 is optionally substituted C6_8 alkyl. In some embodiments, R7 is optionally substituted C6_7 alkyl.
[0160] In some embodiments, R7 is optionally substituted C8_50 alkyl. In some embodiments, R7 is optionally substituted C8_40 alkyl. In some embodiments, R7 is optionally substituted C8_30 alkyl. In some embodiments, R7 is optionally substituted C8_20 alkyl. In some embodiments, R7 is optionally substituted C8_19 alkyl. In some embodiments, R7 is optionally substituted C8_18 alkyl. In some embodiments, R7 is optionally substituted C8_17 alkyl. In some embodiments, R7 is optionally substituted C8_16 alkyl. In some embodiments, R7 is optionally substituted C8_15 alkyl. In some embodiments, R7 is optionally substituted C8_14 alkyl. In some embodiments, R7 is optionally substituted C8_13 alkyl. In some embodiments, R7 is optionally substituted C8_12 alkyl. In some embodiments, R7 is optionally substituted C8_11 alkyl. In some embodiments, R7 is optionally substituted C8_10 alkyl. In some embodiments, R7 is optionally substituted C8_9 alkyl.
[0161] In some embodiments, R7 is optionally substituted C9_50 alkyl. In some embodiments, R7 is optionally substituted C9_40 alkyl. In some embodiments, R7 is optionally substituted C9_30 alkyl. In some embodiments, R7 is optionally substituted C9_20 alkyl. In some embodiments, R7 is optionally substituted C9_19 alkyl. In some embodiments, R7 is optionally substituted C9_18 alkyl. In some embodiments, R7 is optionally substituted C9_17 alkyl. In some embodiments, R7 is optionally substituted C9_16 alkyl. In some embodiments, R7 is optionally substituted C9_15 alkyl. In some embodiments, R7 is optionally substituted C9_14 alkyl. In some embodiments, R7 is optionally substituted C9_13 alkyl. In some embodiments, R7 is optionally substituted C9_12 alkyl. In some embodiments, R7 is optionally substituted C9_11 alkyl. In some embodiments, R7 is optionally substituted C9_10 alkyl.
[0162] In some embodiments, R7 is optionally substituted C100 alkyl. In some embodiments, R7 is optionally substituted Cio-4o alkyl. In some embodiments, R7 is optionally substituted Cio_30 alkyl. In some embodiments, R7 is optionally substituted C1o_20 alkyl. In some embodiments, R7 is optionally substituted C10-19 alkyl. In some embodiments, R7 is optionally substituted Cio_ig alkyl. In some embodiments, R7 is optionally substituted C1o_17 alkyl. In some embodiments, R7 is optionally substituted Cio-16 alkyl. In some embodiments, R7 is optionally substituted C10_15 alkyl. In some embodiments, R7 is optionally substituted C1o_14 alkyl. In some embodiments, R7 is optionally substituted C10-13 alkyl. In some embodiments, R7 is optionally substituted Cio_12 alkyl. In some embodiments, R7 is optionally substituted C10_11 alkyl.
[0163] In some embodiments, R7 is optionally substituted C11-50 alkyl. In some embodiments, R7 is optionally substituted C 1 1-4o alkyl. In some embodiments, R7 is optionally substituted Ci1_30 alkyl. In some embodiments, R7 is optionally substituted Ci1_20 alkyl. In some embodiments, R7 is optionally substituted C11-19 alkyl. In some embodiments, R7 is optionally substituted C1118 alkyl. In some embodiments, R7 is optionally substituted Ci1_17 alkyl. In some embodiments, R7 is optionally substituted CH-16 alkyl. In some embodiments, R7 is optionally substituted Cii_i5 alkyl. In some embodiments, R7 is optionally substituted C11_14 alkyl. In some embodiments, R7 is optionally substituted C11-13 alkyl. In some embodiments, R7 is optionally substituted C11-12 alkyl.
[0164] In some embodiments, R7 is optionally substituted C12_50 alkyl. In some embodiments, R7 is optionally substituted C12-40 alkyl. In some embodiments, R7 is optionally substituted C12_30 alkyl. In some embodiments, R7 is optionally substituted C12_20 alkyl. In some embodiments, R7 is optionally substituted C12-19 alkyl. In some embodiments, R7 is optionally substituted C12_18 alkyl. In some embodiments, R7 is optionally substituted C12_17 alkyl. In some embodiments, R7 is optionally substituted C12-16 alkyl. In some embodiments, R7 is optionally substituted C12_15 alkyl. In some embodiments, R7 is optionally substituted C12_14 alkyl. In some embodiments, R7 is optionally substituted C1243 alkyl.
[0165] In some embodiments, R7 is optionally substituted C6 alkyl. In some embodiments, R7 is optionally substituted C7 alkyl. In some embodiments, R7 is optionally substituted C8 alkyl. In some embodiments, R7 is optionally substituted C9 alkyl. In some embodiments, R7 is optionally substituted C10 alkyl. In some embodiments, R7 is optionally substituted C 1 1 alkyl. In some embodiments, R7 is optionally substituted C12 alkyl. In some embodiments, R7 is optionally substituted C13 alkyl. In some embodiments, R7 is optionally substituted C14 alkyl. In some embodiments, R7 is optionally substituted C15 alkyl. In some embodiments, R7 is optionally substituted C16 alkyl. In some embodiments, R7 is optionally substituted C17 alkyl. In some embodiments, R7 is optionally substituted C18 alkyl. In some embodiments, R7 is optionally substituted C19 alkyl. In some embodiments, R7 is optionally substituted C20 alkyl.
[0166] In some embodiments, for example, in any of the above embodiments, R7 is a substituted alkyl group. In some embodiments, R7 is an unsubstituted alkyl group. In some embodiments, R7 is an optionally substituted straight-chain alkyl group. In some embodiments, R7 is a substituted straight-chain alkyl group. In some embodiments, R7 is an unsubstituted straight-chain alkyl group. In some embodiments, R7 is an optionally substituted branched alkyl group. In some embodiments, R7 is a substituted branched alkyl group. In some embodiments, R7 is an unsubstituted branched alkyl group.
[0167] In some embodiments, R7 is optionally substituted alkenyl. In some embodiments, R7 is optionally substituted C2_50 alkenyl. In some embodiments, R7 is optionally substituted C2_40 alkenyl. In some embodiments, R7 is optionally substituted C2_30 alkenyl. In some embodiments, R7 is optionally substituted C2_20 alkenyl. In some embodiments, R7 is optionally substituted C2_19 alkenyl. In some embodiments, R7 is optionally substituted C2-18 alkenyl. In some embodiments, R7 is optionally substituted C2_17 alkenyl. In some embodiments, R7 is optionally substituted C2_16 alkenyl. In some embodiments, R7 is optionally substituted C2_ 15 alkenyl. In some embodiments, R7 is optionally substituted C2_14 alkenyl.
In some embodiments, R7 is optionally substituted C2_13 alkenyl. In some embodiments, R7 is optionally substituted C2-12 alkenyl. In some embodiments, R7 is optionally substituted C2_11 alkenyl. In some embodiments, R7 is optionally substituted C2_10 alkenyl. In some embodiments, R7 is optionally substituted C2_9 alkenyl. In some embodiments, R7 is optionally substituted C2-8 alkenyl. In some embodiments, R7 is optionally substituted C2_7 alkenyl. In some embodiments, R7 is optionally substituted C2_6 alkenyl.
[0168] In some embodiments, R7 is optionally substituted C4_50 alkenyl.
In some embodiments, R7 is optionally substituted C4_40 alkenyl. In some embodiments, R7 is optionally substituted C4-30 alkenyl. In some embodiments, R7 is optionally substituted C4_20 alkenyl. In some embodiments, R7 is optionally substituted C4_19 alkenyl. In some embodiments, R7 is optionally substituted C4_18 alkenyl. In some embodiments, R7 is optionally substituted C4-17 alkenyl. In some embodiments, R7 is optionally substituted C4_16 alkenyl. In some embodiments, R7 is optionally substituted C4_15 alkenyl. In some embodiments, R7 is optionally substituted C4_ 14 alkenyl. In some embodiments, R7 is optionally substituted C4_13 alkenyl.
In some embodiments, R7 is optionally substituted C4_12 alkenyl. In some embodiments, R7 is optionally substituted C4_11 alkenyl. In some embodiments, R7 is optionally substituted C4_10 alkenyl. In some embodiments, R7 is optionally substituted C4_9 alkenyl. In some embodiments, R7 is optionally substituted C4_8 alkenyl. In some embodiments, R7 is optionally substituted C4-7 alkenyl. In some embodiments, R7 is optionally substituted C4_6 alkenyl.

IS
`siuoulTpoqulo mos ui .pcuo3HE 91-6D polmpscins iCupuopdo ST Li `siuoulTpoqulo mos ui liCuo3HE
Li-oD polmpscins iCipuopdo ST z,i `siuoulTpoqulo aims ui .pcu03HE 8 1-6D
polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui 1Jcuo3HE 61-6D polmpscins iCipuopdo ST LI' `sluoulTpoqulo mos uI .1,Cuo3IIP oz-oD polmpscins iCipuopdo ST Li `siuoulTpoqulo mos ui .pcu03up 9E-6D polmpscins ictpuopdo ST LI' `sluoulTpoqulo mos ui 1icuo3up 0t-6D polmpscins iCipuopdo ST
LI' `siuoulTpoqulo mos ui .pcu03up 0s-6D polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui IILIO]
licuolip 6-8D polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos uI *IiCu03IIP o-r-sD polmpscins iCipuopdo ST Li `siuoulTpoqulo mos ui .pcu03up II-8D polmpscins iCupuopdo ST Li `siuoulTpoqulo mos ui 1iCuo3up zI-8D polmpsqns iCipuopdo ST
Li `siuoulTpoqulo mos ui liCu03up EI-8D polmpsqns iCupuopdo ST z,i `siuoulTpoqulo mos ui liCu03up tI
-8D polmpsqns iCipuopdo ST Li `siuoulTpoqulo mos ui 1iCuo3up cI-8D pompsqns iCipuopdo sl. Li `siuoulTpoqulo mos ui 11Cuo3up 91-8D polmpsqns iCupuopdo ST Li `siuoulTpoqulo mos ui 11Cuo3up LTD polmpscins iCipuopdo ST z,i `siuoulTpoqulo mos ui 1Jcuo3up 8 1-8D
polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui .pcu03up 61-8D polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos uI *IiCuo3IIP oz-sD polmpscins iCipuopdo ST Li `siuoulTpoqulo mos ui .pcu03up 0E-8D polmpscins ictpuopdo ST LI' `siuoulTpoqulo mos ui 1icuo3up 0i7-8D polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui 1iCuo3up 9c-8D polmpsqns iCipuopdo ST z,i `siuoulTpoqulo mos ui [OLIO]
.pcuo3up L-9D polmpscins iCipuopdo ST Li `siuoulTpoqulo mos ui 1Jcuo3up 8-9D
polmpscins iCipuopdo ST z,i `siuoulTpoqulo mos ui 1iCuo3up 6-9D polmpsqns iCipuopdo ST z,i `siuoulTpoqulo mos uI *IiCuo3IIP o1-9D polmpscins iCipuopdo ST Li `siuoulTpoqulo mos ui .pcu03up II-9D polmpscins iCupuopdo ST Li `siuoulTpoqulo mos ui 1iCuo3up zI-9D polmpsqns iCipuopdo ST
Li `siuoulTpoqulo mos ui 1iCuo3up EI-9D polmpsqns iCupuopdo ST z,i `siuoulTpoqulo mos ui 1iCuo3up tI
-9D polmpsqns iCipuopdo ST Li `siuoulTpoqulo mos ui 1iCuo3up cI-9D pompsqns iCipuopdo ST Li `siuoulTpoqulo mos ui 1iCuo3up 9I-9D polmpsqns iCupuopdo ST Li `siuoulTpoqulo mos ui 1iCuo3up LI-9D polmpscins iCipuopdo ST z,i `siuoulTpoqulo mos ui 1Jcuo3up 8 1-9D
polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui 1Jcuo3up 61-9D polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos uI *IiCuo3IIP oz-9D polmpscins iCipuopdo ST Li `siuoulTpoqulo mos ui .pcu03up 0E-9D polmpscins iCupuopdo ST Li `siuoulTpoqulo mos ui 1iCuo3up 9t-9D polmpsqns iCipuopdo ST
Li `siuoulTpoqulo mos ui 1Jcuo3up 0s-9D polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui 16910]
6LI90/tIOZSI1IIDd L9tI90/SIOZ OM

ZS
alms ui =pCuo3HE 9i-zip polmpsqns iCupuopdo ST Li `siuoulTpoqulo aims ui =pCuo3HE
Li-ziD palmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui =pcuo3HE si-ziD
palmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui Itc1103HE 6I -Zip polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos uI .1,Cuo3IIP oz-ziD pompscins iCupuopdo ST LI' `siuoulTpoqulo mos ui =pcu03up oE-ziD polmpscins icipuopdo ST LI' `siuoulTpoqulo mos ui 1Jcuo3up ot-ziD palmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui =pcu03up os-zID palmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui Iraol lictio3up zi-TID polmpsqns iCipuopdo ST z,i `siuoulTpoqulo mos uI *IiCu03IIP ET-T ID pompsqns iCupuopdo ST z,i `siuoulTpoqulo mos ui =pCu03up 17TT ID polmpsqns iCipuopdo ST Li `siuoulTpoqulo mos ui 1JCuo3up cI-HD polmpsqns iCipuopdo ST
Li `siuoulTpoqulo mos ui =pCu03up 9111D polmpsqns iCupuopdo ST Li `siuoulTpoqulo mos ui =pCu03up Li-i ID polmpscins iCipuopdo ST LI' `siuoulTpoqulo aims ui =pcuo3up si-i ID
polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui Itc1103up 61-IID polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos uI *IiCu03IIP oz-i ID pompscins iCupuopdo ST LI' `siuoulTpoqulo mos ui =pcu03up 0-I ID polmpscins icipuopdo ST LI' `siuoulTpoqulo mos ui 1Jcuo3up ot-iiD palmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui 1Jcuo3up os-i ID palmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui [CLIO]
1Jcuo3up 11-0ID polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui 1Jcuo3up zi-oiD palmpscins iCipuopdo ST LI' `siuoulTpoqulo mos uI *IiCuo3IIP I-OID pompscins iCupuopdo ST LI' `siuoulTpoqulo mos ui 1Jcuo3up t-r-oID polmpscins icipuopdo ST LI' `siuoulTpoqulo mos ui 1Jcuo3up sT-oID palmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui 1Jcuo3up 91 'D polmpscins iCupuopdo ST LI' `siuoulTpoqulo mos ui 1JCuo3up Li-oiD palmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui =pcuo3up si-oiD
palmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui 1Jcuo3up 61-oiD palmpscins iCipuopdo ST LI' `siuoulTpoqulo mos uI *IiCuo3IIP oz-oID pompscins iCupuopdo ST LI' `siuoulTpoqulo mos ui 1Jcuo3up oE-oID polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui 1Jcuo3up ot-oID polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui 1Jcuo3up os-oID palmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui IZLIO]
=pcuolip 01-6D polmpscins iCipuopdo ST Li `siuoulTpoqulo mos ui =pcuo3up I1-6D polmpscins ictpuopdo ST LI' `siuoulTpoqulo mos ui 1Jcuo3up zI-6D polmpscins iCipuopdo ST
LI' `siuoulTpoqulo alms ui 1JCuo3up EI-6D polmpscins iCupuopdo ST LI' `siuoulTpoqulo alms ui 1JCuo3up ti -6D polmRscins icipuopdo ST LI' `siuoulTpoqulo alms ui 1Jcuo3up ci-6D
pompscins icipuopdo ST LI' 6LI90/tIOZSI1IIDd L9tI90/SIOZ OM

embodiments, R7 is optionally substituted C12-15 alkenyl. In some embodiments, R7 is optionally substituted C12_14 alkenyl. In some embodiments, R7 is optionally substituted C12_13 alkenyl.
[0175] In some embodiments, R7 is optionally substituted C6 alkenyl. In some embodiments, R7 is optionally substituted C7 alkenyl. In some embodiments, R7 is optionally substituted C8 alkenyl. In some embodiments, R7 is optionally substituted C9 alkenyl. In some embodiments, R7 is optionally substituted C10 alkenyl. In some embodiments, R7 is optionally substituted Cii alkenyl. In some embodiments, R7 is optionally substituted C12 alkenyl. In some embodiments, R7 is optionally substituted C13 alkenyl. In some embodiments, R7 is optionally substituted C14 alkenyl. In some embodiments, R7 is optionally substituted C15 alkenyl. In some embodiments, R7 is optionally substituted C16 alkenyl. In some embodiments, R7 is optionally substituted C17 alkenyl. In some embodiments, R7 is optionally substituted C18 alkenyl. In some embodiments, R7 is optionally substituted C19 alkenyl. In some embodiments, R7 is optionally substituted C20 alkenyl.
[0176] In some embodiments, for example, in any of the above embodiments, R7 is a substituted alkenyl group. In some embodiments, R7 is an unsubstituted alkenyl group. In some embodiments, R7 is an optionally substituted straight-chain alkenyl group. In some embodiments, R7 is a substituted straight-chain alkenyl group. In some embodiments, R7 is an unsubstituted straight-chain alkenyl group. In some embodiments, R7 is an optionally substituted branched alkenyl group. In some embodiments, R7 is a substituted branched alkenyl group. In some embodiments, R7 is an unsubstituted branched alkenyl group.
[0177] In some embodiments, R7 is optionally substituted alkynyl. In some embodiments, R7 is optionally substituted C2_50 alkynyl. In some embodiments, R7 is optionally substituted C2_40 alkynyl. In some embodiments, R7 is optionally substituted C2_30 alkynyl. In some embodiments, R7 is optionally substituted C2_20 alkynyl. In some embodiments, R7 is optionally substituted C2_19 alkynyl. In some embodiments, R7 is optionally substituted C2_18 alkynyl. In some embodiments, R7 is optionally substituted C2_17 alkynyl. In some embodiments, R7 is optionally substituted C2_16 alkynyl. In some embodiments, R7 is optionally substituted C2_15 alkynyl. In some embodiments, R7 is optionally substituted C2_14 alkynyl. In some embodiments, R7 is optionally substituted C2_13 alkynyl. In some embodiments, R7 is optionally substituted C2_12 alkynyl. In some embodiments, R7 is optionally substituted C2_11 alkynyl. In some embodiments, R7 is optionally substituted C2_10 alkynyl. In some embodiments, R7 is optionally substituted C2_9 alkynyl. In some embodiments, R7 is optionally substituted C2_8 alkynyl. In some embodiments, R7 is optionally substituted C2_7 alkynyl. In some embodiments, R7 is optionally substituted C2_6 alkynyl.
[0178] In some embodiments, R7 is optionally substituted C4_50 alkynyl.
In some embodiments, R7 is optionally substituted C4_40 alkynyl. In some embodiments, R7 is optionally substituted C4_30 alkynyl. In some embodiments, R7 is optionally substituted C4_20 alkynyl. In some embodiments, R7 is optionally substituted C4_19 alkynyl. In some embodiments, R7 is optionally substituted C4_18 alkynyl. In some embodiments, R7 is optionally substituted C4_17 alkynyl. In some embodiments, R7 is optionally substituted C4_16 alkynyl. In some embodiments, R7 is optionally substituted C4_15 alkynyl. In some embodiments, R7 is optionally substituted C4_14 alkynyl. In some embodiments, R7 is optionally substituted C4_13 alkynyl. In some embodiments, R7 is optionally substituted C4_12 alkynyl. In some embodiments, R7 is optionally substituted C4_11 alkynyl. In some embodiments, R7 is optionally substituted C4_10 alkynyl. In some embodiments, R7 is optionally substituted C4_9 alkynyl. In some embodiments, R7 is optionally substituted C4_8 alkynyl. In some embodiments, R7 is optionally substituted C4_7 alkynyl. In some embodiments, R7 is optionally substituted C4_6 alkynyl.
[0179] In some embodiments, R7 is optionally substituted C6_50 alkynyl.
In some embodiments, R7 is optionally substituted C6_40 alkynyl. In some embodiments, R7 is optionally substituted C6_30 alkynyl. In some embodiments, R7 is optionally substituted C6_20 alkynyl. In some embodiments, R7 is optionally substituted C6_19 alkynyl. In some embodiments, R7 is optionally substituted C6_18 alkynyl. In some embodiments, R7 is optionally substituted C6_17 alkynyl. In some embodiments, R7 is optionally substituted C6_16 alkynyl. In some embodiments, R7 is optionally substituted C6_15 alkynyl. In some embodiments, R7 is optionally substituted C6_14 alkynyl. In some embodiments, R7 is optionally substituted C6_13 alkynyl. In some embodiments, R7 is optionally substituted C6_12 alkynyl. In some embodiments, R7 is optionally substituted C6_11 alkynyl. In some embodiments, R7 is optionally substituted C6_10 alkynyl. In some embodiments, R7 is optionally substituted C6_9 alkynyl. In some embodiments, R7 is optionally substituted C6_8 alkynyl. In some embodiments, R7 is optionally substituted C6_7 alkynyl.

Si UT .IICTIA3TIP ET-OID palmRscins iCipuopdo ST LI' `siuoulTpoqulo aims ui =pcuic3up ti-oiD polmpscins icipuopdo ST LI' `siuoulTpoqulo mos ui 1Jcuic3up sT-oID palmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui licu1c3up 91-oiD palmRscins iCipuopdo ST LI' `siuoulTpoqulo mos ui =pCu1C3up Li-oiD polmpscins iCtpuopdo ST LI' `siuoulTpoqulo mos ui 1Jcuic3up sT-oID
polmpscins iCipuopdo ST II `siuoulTpoqulo mos ui =pcuic3up 61-oiD palmpscins iCipuop L
do ST II `siuoulTpoqulo mos = L =
uI *IiCuiC3IIP oz-oiD palmRscins iCipuopdo ST LI' `siuoulTpoqulo mos ui =pcu1c3up oE-oiD polmpscins icipuopdo ST LI' `siuoulTpoqulo mos ui 1Jcuic3up ot-oiD palmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui =pcu1c3up os-oID polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui [Z810]
=pcuiC3up 01-6D polmpscins iCipuopdo ST LI' `siuoulTpoqulo aims ui liCuic3up H-6D
polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui 1iCuic3up zI-6D polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos uI *IiCu1C3IIP EI-6D polmpscins iCtpuopdo ST LI' `siuoulTpoqulo mos ui liCu1c3up tI-6D polmpscins icipuopdo ST Li `siuoulTpoqulo mos ui 1JCuic3up si-6D polmpscins iCipuopdo ST
LI' `siuoulTpoqulo mos ui liCu1c3up 91-6D polmpscins iCipuopdo ST z,i `siuoulTpoqulo mos ui =pCu1C3up LI-6D polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui liCu1c3up 8 1-6D
polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui liCu1c3up 61-6D polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos uI *IiCu1C3IIP oz-6D polmpscins iCtpuopdo ST LI' `siuoulTpoqulo mos ui liCu1c3up 0E-6D polmpscins icipuopdo ST Li `siuoulTpoqulo mos ui 1JCuic3up ot-6D polmpscins iCipuopdo ST
LI' `siuoulTpoqulo mos ui 1iCuic3up OC-6D polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui [IMO]
1'cuic3up 6-8D polmpscins iCipuopdo ST z,i `siuoulTpoqulo mos ui 1JCuiC3up OT-sp polmpsqns iCipuopdo ST z,i `siuoulTpoqulo mos ui liCu1c3up II-8D
polmpscins iCipuopdo ST Li `siuoulTpoqulo mos ui 1JCuiC3up p polmpsqns iCipuopdo ST z,i `siuoulTpoqulo mos uI *IiCuiC3IIP ET-8D polmpsqns iCupuopdo ST z,i `siuoulTpoqulo mos ui =pCu1C3up p polmpsqns iCipuopdo ST Li `siuoulTpoqulo mos ui 1JCuiC3up cI-8D polmpsqns iCipuopdo ST
Li `siuoulTpoqulo mos ui =1JCuiC3up 91-8D polmpsqns iCipuopdo ST z,i `siuoulTpoqulo mos ui 1JCuiC3up Li-sp polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui liCu1c3up 81-8D
polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui 1iCuic3up 61-8D polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos uI *IiCuiC3IIP oz-sp polmpscins iCtpuopdo ST LI' `siuoulTpoqulo mos ui liCuic3up 0E-8D polmpscins icipuopdo ST Li `siuoulTpoqulo mos ui 1JCuic3up 017-8D polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui 1iCuic3up os-sp polmpscins iCipuopdo ST LI' `siuoulTpoqulo mos ui 10810]
6LI90/tIOZSI1IIDd L9tI90/SIOZ OM

some embodiments, R7 is optionally substituted C10-12 alkynyl. In some embodiments, R7 is optionally substituted C10_11 alkynyl.
[0183] In some embodiments, R7 is optionally substituted C11-50 alkynyl.
In some embodiments, R7 is optionally substituted CH-4o alkynyl. In some embodiments, R7 is optionally substituted Ci1_30 alkynyl. In some embodiments, R7 is optionally substituted C11_20 alkynyl. In some embodiments, R7 is optionally substituted C11-19 alkynyl. In some embodiments, R7 is optionally substituted C11_18 alkynyl. In some embodiments, R7 is optionally substituted C11_17 alkynyl. In some embodiments, R7 is optionally substituted C11-16 alkynyl. In some embodiments, R7 is optionally substituted C11-15 alkynyl. In some embodiments, R7 is optionally substituted C11_14 alkynyl. In some embodiments, R7 is optionally substituted C11_13 alkynyl. In some embodiments, R7 is optionally substituted C11-12 alkynyl.
[0184] In some embodiments, R7 is optionally substituted C12_50 alkynyl.
In some embodiments, R7 is optionally substituted C12-4o alkynyl. In some embodiments, R7 is optionally substituted C12_30 alkynyl. In some embodiments, R7 is optionally substituted C12_20 alkynyl. In some embodiments, R7 is optionally substituted C12-19 alkynyl. In some embodiments, R7 is optionally substituted C12_18 alkynyl. In some embodiments, R7 is optionally substituted C12_17 alkynyl. In some embodiments, R7 is optionally substituted C12-16 alkynyl. In some embodiments, R7 is optionally substituted C12-15 alkynyl. In some embodiments, R7 is optionally substituted C1214 alkynyl. In some embodiments, R7 is optionally substituted C12_13 alkynyl.
[0185] In some embodiments, R7 is optionally substituted C6 alkynyl. In some embodiments, R7 is optionally substituted C7 alkynyl. In some embodiments, R7 is optionally substituted C8 alkynyl. In some embodiments, R7 is optionally substituted C9 alkynyl. In some embodiments, R7 is optionally substituted C10 alkynyl. In some embodiments, R7 is optionally substituted Cii alkynyl. In some embodiments, R7 is optionally substituted C12 alkynyl. In some embodiments, R7 is optionally substituted C13 alkynyl. In some embodiments, R7 is optionally substituted Ci4 alkynyl. In some embodiments, R7 is optionally substituted C15 alkynyl. In some embodiments, R7 is optionally substituted C16 alkynyl. In some embodiments, R7 is optionally substituted Ci7 alkynyl. In some embodiments, R7 is optionally substituted C18 alkynyl. In some embodiments, R7 is optionally substituted Ci9 alkynyl. In some embodiments, R7 is optionally substituted C20 alkynyl.

[0186] In some embodiments, for example, in any of the above embodiments, R7 is a substituted alkynyl group. In some embodiments, R7 is an unsubstituted alkynyl group. In some embodiments, R7 is an optionally substituted straight-chain alkynyl group. In some embodiments, R7 is a substituted straight-chain alkynyl group. In some embodiments, R7 is an unsubstituted straight-chain alkynyl group. In some embodiments, R7 is an optionally substituted branched alkynyl group. In some embodiments, R7 is a substituted branched alkynyl group. In some embodiments, R7 is an unsubstituted branched alkynyl group.
[0187] In some embodiments, R7 is optionally substituted carbocyclyl. In some embodiments, R7 is optionally substituted heterocyclyl. In some embodiments, R7 is optionally substituted aryl. In some embodiments, R7 is optionally substituted heteroaryl. In some embodiments, R7 is a nitrogen protecting group.
[0188] In some embodiments, R7 is a group of formula (i). In some embodiments, R7 is a RL
group of formula (i-a). In some embodiments, R7 is a group of formula ¨1 (i-al). In some embodiments, R7 is a group of formula (i-b). In some embodiments, R7 is a group of formula (ii). In some embodiments, R7 is a group of formula (iii).
[0189] In some embodiments, at least one instance of R6 and R7 is a group of the formula (i), (ii) or (iii). In some embodiments, each instance of R6 and R7 is independently a group of the formula (i), (ii) or (iii). In some embodiments, each instance of R6 and R7 is independently a group of the formula (i). In some embodiments, each instance of R6 and R7 is independently a group of the formula (i-a). In some embodiments, each instance of R6 and R7 is independently a group of the formula (i-b). In some embodiments, each instance of R6 and R7 is independently a group of the formula (ii). In some embodiments, each instance of R6 and R7 is independently a group of the formula (iii).
[0190] In some embodiments, R6 and R7 are the same. In some embodiments, R6 and R7 are different.
[0191] In certain embodiments, both R6 and R7 are hydrogen. In certain embodiments, R6 is hydrogen and R7 is a group of the formula (i), (ii), or (iii). In certain embodiments, R6 is hydrogen and R7 is a group of the formula (i). In certain embodiments, R6 is hydrogen and R7 is a group of the formula (ii). In certain embodiments, R6 is hydrogen and R7 is a group of the formula (iii). In certain embodiments, each of R6 and R7 is independently a group of the formula (i), (ii), or (iii). In certain embodiments, each of R6 and R7 is independently a group of the formula (i). In certain embodiments, each of R6 and R7 is independently a group of the formula (ii). In certain embodiments, each of R6 and R7 is independently a group of the formula (iii). In certain embodiments, R6 and R7 are the same group, which is selected from formulas (i), (ii), and (iii). In some embodiments, R6 and R7 are the same group of formula (i). In some embodiments, R6 and R7 are the same group of formula (i-a). In some embodiments, R6 and R7 are the same group of formula (i-al). In some embodiments, R6 and R7 are the same group of formula (i-b).
RL
[0192] In some embodiments, R6 and R7 are the same group of formula ¨1 wherein RL is as defined above and described herein. In some embodiments, R6 and R7 are the RL
same group of formula A (i-al), wherein RL is optionally substituted Ci_50alkyl, optionally substituted C2_50alkenyl, optionally substituted C2_50alkynyl, optionally substituted heteroCi_50alkyl, optionally substituted heteroC2_50alkenyl, or optionally substituted heteroC2_ RL
soalkynyl. In some embodiments, R6 and R7 are the same group of formula A
(i-al), wherein RL is optionally substituted C5_50a1ky1, optionally substituted C5_50alkenyl, optionally substituted C5_50alkynyl, optionally substituted heteroC5_50alkyl, optionally substituted heteroC5_ 50alkenyl, or optionally substituted heteroC5_50alkynyl. In some embodiments, R6 and R7 are the RL
same group of formula A (i-al), wherein RL is optionally substituted C5_40a1ky1, optionally substituted C5_40alkenyl, optionally substituted C5_40alkynyl, optionally substituted heteroC5_40alkyl, optionally substituted heteroC5_40alkenyl, or optionally substituted heteroC5_ RL
4oalkynyl. In some embodiments, R6 and R7 are the same group of formula A
(i-al), wherein RL is optionally substituted C5_30a1ky1, optionally substituted C5_30alkenyl, optionally substituted C5_30alkynyl, optionally substituted heteroC5_30alkyl, optionally substituted heteroC5_ 30alkenyl, or optionally substituted heteroC5_30alkynyl. In some embodiments, R6 and R7 are the RL
)-0H
same group of formula A (i-al), wherein RL is optionally substituted C5_25alkyl, optionally substituted C5_25alkenyl, optionally substituted C5_25alkynyl, optionally substituted heteroC5_25alkyl, optionally substituted heteroC5_25alkenyl, or optionally substituted heteroC5_ RL
_)--OH
25alkynyl. In some embodiments, R6 and R7 are the same group of formula A
(i-al), wherein RL is optionally substituted C5_20alkyl, optionally substituted C5_20alkenyl, optionally substituted C5_20alkynyl, optionally substituted heteroC5_20alkyl, optionally substituted heteroC5_ 20alkenyl, or optionally substituted heteroC5_20alkynyl. In some embodiments, R6 and R7 are the RL
_)--OH
same group of formula A (i-al), wherein RL is optionally substituted C5_15alkyl, optionally substituted C5_15alkenyl, optionally substituted C5_15alkynyl, optionally substituted heteroC5_15alkyl, optionally substituted heteroC5_15alkenyl, or optionally substituted heteroC5_ RL
)¨OH
isalkynyl. In some embodiments, R6 and R7 are the same group of formula A
(i-al), wherein RL is optionally substituted C5 alkyl, optionally substituted C5 alkenyl, optionally substituted C5 alkynyl, optionally substituted heteroC5alkyl, optionally substituted heteroC5alkenyl, or optionally substituted heteroC5alkynyl. In some embodiments, R6 and R7 are RL
)--OH
the same group of formula A (i-a I), wherein RL is optionally substituted C6 alkyl, optionally substituted C6 alkenyl, optionally substituted C6 alkynyl, optionally substituted heteroC6alkyl, optionally substituted heteroC6alkenyl, or optionally substituted heteroC6alkynyl.
RL
_)--OH
In some embodiments, R6 and R7 are the same group of formula A (i-al), wherein RL is optionally substituted C7 alkyl, optionally substituted C7 alkenyl, optionally substituted C7 alkynyl, optionally substituted heteroC7alkyl, optionally substituted heteroC7alkenyl, or optionally substituted heteroC7alkynyl. In some embodiments, R6 and R7 are the same group of RL
)-0H
formula A
(i-al), wherein RL is optionally substituted C8 alkyl, optionally substituted alkenyl, optionally substituted C8 alkynyl, optionally substituted heteroC8alkyl, optionally substituted heteroC8alkenyl, or optionally substituted heteroC8alkynyl. In some embodiments, RL
)--OH
R6 and R7 are the same group of formula A (i-al), wherein RL is optionally substituted C9 alkyl, optionally substituted C9 alkenyl, optionally substituted C9 alkynyl, optionally substituted heteroC9alkyl, optionally substituted heteroC9alkenyl, or optionally substituted RL
_)--OH
heteroC9alkynyl. In some embodiments, R6 and R7 are the same group of formula A (i-al), wherein RL is optionally substituted C10 alkyl, optionally substituted C
10 alkenyl, optionally substituted Ci0 alkynyl, optionally substituted heteroCi0alkyl, optionally substituted heteroCi0alkenyl, or optionally substituted heteroCi0alkynyl. In some embodiments, R6 and R7 RL
I-OH
are the same group of formula A (i-al), wherein RL is optionally substituted C11 alkyl, optionally substituted Cii alkenyl, optionally substituted C11 alkynyl, optionally substituted heteroCiialkyl, optionally substituted heteroCiialkenyl, or optionally substituted RL
_)--OH
heteroC 1 ialkynyl. In some embodiments, R6 and R7 are the same group of formula A (i-al), wherein RL is optionally substituted C12 alkyl, optionally substituted C12 alkenyl, optionally substituted C12 alkynyl, optionally substituted heteroC ualkyl, optionally substituted heteroC ualkenyl, or optionally substituted heteroC ualkynyl. In some embodiments, R6 and R7 RL
_)--OH
are the same group of formula A (i-al), wherein RL is optionally substituted C13 alkyl, optionally substituted C13 alkenyl, optionally substituted C 13 alkynyl, optionally substituted heteroC nalkyl, optionally substituted heteroCnalkenyl, or optionally substituted RL
)-OH
heteroCnalkynyl. In some embodiments, R6 and R7 are the same group of formula ¨1 (i-al), wherein RL is optionally substituted C14 alkyl, optionally substituted C14 alkenyl, optionally substituted C14 alkynyl, optionally substituted heteroCmalkyl, optionally substituted heteroCmalkenyl, or optionally substituted heteroCmalkynyl. In some embodiments, R6 and R7 RL
)-0H
are the same group of formula ¨1 (i-al), wherein RL is optionally substituted C15 alkyl, optionally substituted C15 alkenyl, optionally substituted C15 alkynyl, optionally substituted heteroC 15 alkyl, optionally substituted heteroCi5alkenyl, or optionally substituted heteroC 15 alkynyl.
RL
)¨OH
[0193] In some embodiments, R6 and R7 are the same group of formula ¨1 (i-al), wherein RL is as defined above and described herein. In some embodiments, R6 and R7 are the RL
)-0H
same group of formula ¨1 (i-al), wherein RL is optionally substituted Ci_50alkyl. In some RL
) _______________________________________________ OH
embodiments, R6 and R7 are the same group of formula ¨1 (i-al), wherein RL
is optionally substituted C5_50alkyl. In some embodiments, R6 and R7 are the same group of RL
_)--OH
formula ¨1 (i-al), wherein RL is optionally substituted C5_40alkyl. In some embodiments, RL
_)--OH
R6 and R7 are the same group of formula ¨1 (i-al), wherein RL is optionally substituted RL
)¨OH
C5_30a1ky1. In some embodiments, R6 and R7 are the same group of formula ¨1 (i-al), wherein RL is optionally substituted C5_25a1ky1. In some embodiments, R6 and R7 are the same RL
)-0H
group of formula A (i-al), wherein RL is optionally substituted C5_20a1ky1.
In some RL
_)--OH
embodiments, R6 and R7 are the same group of formula A (i-al), wherein RL
is optionally substituted C5_15alkyl. In some embodiments, R6 and R7 are the same group of RL
_)--OH
formula A (i-al), wherein RL is optionally substituted C5 alkyl. In some embodiments, RL
)--OH
R6 and R7 are the same group of formula A (i-al), wherein RL is optionally substituted RL
_)--OH
C6 alkyl. In some embodiments, R6 and R7 are the same group of formula A (i-a 1), wherein RL is optionally substituted C7 alkyl. In some embodiments, R6 and R7 are the same RL
)--OH
group of formula A (i-al), wherein RL is optionally substituted C8 alkyl.
In some RL
_)--OH
embodiments, R6 and R7 are the same group of formula A (i-al), wherein RL
is optionally substituted C9 alkyl. In some embodiments, R6 and R7 are the same group of formula RL
)--OH
-1= L =
(i-al), wherein R is optionally substituted C10 alkyl. In some embodiments, R6 and RL
)--OH
= =
R7 are the same group of formula A (i-al), wherein RL is optionally substituted Cii alkyl.
RL
_)--OH
In some embodiments, R6 and R7 are the same group of formula A (i-al), wherein RL is optionally substituted C12 alkyl. In some embodiments, R6 and R7 are the same group of formula RL
-1= L =
(i-al), wherein R is optionally substituted C13 alkyl. In some embodiments, R6 and RL
)-OH
= =
R7 are the same group of formula ¨1 (i-al), wherein RL is optionally substituted C14 alkyl.
RL
_)--OH
In some embodiments, R6 and R7 are the same group of formula ¨1 (i-al), wherein RI- is optionally substituted C15 alkyl.
[0194]Al i As generally defined above, each occurrence of R s independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to an sulfur atom, a nitrogen protecting group when attached to a nitrogen atom, or two RA1 groups, together with the nitrogen atom to which they are attached, are joined to form an optionally substituted heterocyclic or optionally substituted heteroaryl ring.
[0195] In some embodiments, RA1 is hydrogen. In some embodiments, RA1 is optionally substituted alkyl. In some embodiments, RA1 is optionally substituted alkenyl.
In some embodiments, RA1 is optionally substituted alkynyl. In some embodiments, RA1 is optionally substituted carbocyclyl. In some embodiments, RA1 is optionally substituted heterocyclyl. In some embodiments, RA1 is optionally substituted aryl. In some embodiments, RA1 is optionally substituted heteroaryl. In some embodiments, RA1 is an oxygen protecting group when attached to an oxygen atom. In some embodiments, RA1 is a sulfur protecting group when attached to a sulfur atom. In some embodiments, RA1 is a nitrogen protecting group when attached to a nitrogen atom. In some embodiments, two RA1 groups, together with the nitrogen atom to which they are attached, are joined to form an optionally substituted heterocyclic or optionally substituted heteroaryl ring.
[0196]2 i As generally defined above, each instance of R s independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group, or a group of the formula (i), (ii), or (iii):

R'\ R' XRL
RL )--yRP _______________________________ \ RL

R' R' R
(i) (ii) (iii) wherein each of R', Y, RP, RI- and X is independently as defined above and described herein.
[0197] In some embodiments, R2 is hydrogen. In some embodiments, at least one instance of R2 is hydrogen. In some embodiments, each instance of R2 is hydrogen.
[0198] In certain embodiments, R2 is optionally substituted alkyl; e.g., optionally substituted Ci_6alkyl, optionally substituted C2_6a1ky1, optionally substituted C3_6a1ky1, optionally substituted C4_6a1ky1, optionally substituted C4_5a1ky1, or optionally substituted C3_4a1ky1. In certain embodiments, at least one instance of R2 is optionally substituted alkyl; e.g., optionally substituted Ci_6alkyl, optionally substituted C2_6a1ky1, optionally substituted C3_6a1ky1, optionally substituted C4_6a1ky1, optionally substituted C4_5a1ky1, or optionally substituted C3_4a1ky1.
[0199] In certain embodiments, R2 is optionally substituted alkenyl, e.g., optionally substituted C2_6alkenyl, optionally substituted C3_6alkenyl, optionally substituted C4_6alkenyl, optionally substituted C4_5alkenyl, or optionally substituted C3_4alkenyl. In certain embodiments, at least one instance of R2 is optionally substituted alkenyl, e.g., optionally substituted C2-6alkenyl, optionally substituted C3_6alkenyl, optionally substituted C4_6alkenyl, optionally substituted C4_5alkenyl, or optionally substituted C3_4alkenyl.
[0200] In certain embodiments, R2 is optionally substituted alkynyl, e.g., optionally substituted C2_6alkynyl, optionally substituted C3_6alkynyl, optionally substituted C4_6alkynyl, optionally substituted C4_5alkynyl, or optionally substituted C3_4alkynyl. In certain embodiments, at least one instance of R2 is optionally substituted alkynyl, e.g., optionally substituted C2_ 6alkynyl, optionally substituted C3_6alkynyl, optionally substituted C4_6alkynyl, optionally substituted C4_5alkynyl, or optionally substituted C3_4alkynyl.
[0201] In certain embodiments, R2 is optionally substituted carbocyclyl, e.g., optionally substituted C3_10carbocyclyl, optionally substituted C5_8carbocyclyl, optionally substituted C5_ 6carbocyclyl, optionally substituted C5 carbocyclyl, or optionally substituted C6 carbocyclyl. In certain embodiments, at least one instance of R2 is optionally substituted carbocyclyl, e.g., optionally substituted C3_10carbocyclyl, optionally substituted C5_8carbocyclyl, optionally substituted C5_6carbocyclyl, optionally substituted C5 carbocyclyl, or optionally substituted C6 carbocyclyl.
[0202] In certain embodiments, R2 is optionally substituted heterocyclyl, e.g., optionally substituted 3-14 membered heterocyclyl, optionally substituted 3-10 membered heterocyclyl, optionally substituted 5-8 membered heterocyclyl, optionally substituted 5-6 membered heterocyclyl, optionally substituted 5-membered heterocyclyl, or optionally substituted 6-membered heterocyclyl. In certain embodiments, at least one instance of R2 is optionally substituted heterocyclyl, e.g., optionally substituted 3-14 membered heterocyclyl, optionally substituted 3-10 membered heterocyclyl, optionally substituted 5-8 membered heterocyclyl, optionally substituted 5-6 membered heterocyclyl, optionally substituted 5-membered heterocyclyl, or optionally substituted 6-membered heterocyclyl.
[0203] In certain embodiments, R2 is optionally substituted aryl, e.g., optionally substituted phenyl. In some embodiments, R2 isoptionally substituted phenyl.
In some embodiments, R2 issubstituted phenyl. In some embodiments, R2 isunsubstituted phenyl. In certain embodiments, at least one instance of R2 is optionally substituted aryl, e.g., optionally substituted phenyl. In some embodiments, at least one instance of R2 isoptionally substituted phenyl. In some embodiments, at least one instance of R2 issubstituted phenyl.
In some embodiments, at least one instance of R2 isunsubstituted phenyl.
[0204] In certain embodiments, R2 is optionally substituted heteroaryl, e.g., optionally substituted 5-14 membered heteroaryl, optionally substituted 5-10 membered heteroaryl, optionally substituted 5-6 membered heteroaryl, optionally substituted 5-membered heteroaryl, or optionally substituted 6-membered heteroaryl. In certain embodiments, at least one instance of R2 is optionally substituted heteroaryl, e.g., optionally substituted 5-14 membered heteroaryl, optionally substituted 5-10 membered heteroaryl, optionally substituted 5-6 membered heteroaryl, optionally substituted 5-membered heteroaryl, or optionally substituted 6-membered heteroaryl.

[0205] In some embodiments, R2 is a nitrogen protecting group. In some embodiments, at least one R2 is a nitrogen protecting group.
[0206] In certain embodiments, R2 is a group of the formula (i). In certain embodiments, R2 is a group of the formula (ii). In certain embodiments, R2 is a group of the formula (iii). In certain embodiments, at least one instance of R2 is a group of the formula (i). In certain embodiments, at least one instance of R2 is a group of the formula (ii). In certain embodiments, at least one instance of R2 is a group of the formula (iii).
[0207] In certain embodiments, each instance of R2 is a group other than formula (i), (ii), or (iii); in that instance, it follows that at least one RQ is a group of the formula (i), (ii), or (iii), or at least one Rl is a group of formula (iv), and at least one of R6 or R7 encompassed by Rl is a group of the formula (i), (ii), or (iii). For example, in certain embodiments, both instances of R2 are hydrogen, and thus at least one RQ is a group of the formula (i), (ii), or (iii), or at least one Rl is a group of formula (iv), and at least one of R6 or R7 encompassed by Rl is a group of the formula (i), (ii), or (iii).
[0208] As generally defined above, each instance of R' is independently hydrogen or optionally substituted alkyl. In some embodiments, R' is hydrogen. In some embodiments, R' is substituted alkyl. In certain embodiments, at least one instance of R' is hydrogen. In certain embodiments, at least two instances of R' is hydrogen. In certain embodiments, each instance of R' is hydrogen. In certain embodiments, at least one instance of R' is optionally substituted alkyl, e.g., methyl. In certain embodiments, at least two instances of R' is optionally substituted alkyl, e.g., methyl. In some embodiments, at least one instance of R' is hydrogen, and at least one instance of R' is optionally substituted alkyl. In certain embodiments, one instance of R' is optionally substituted alkyl, and the rest are hydrogen.
[0209] As generally defined above, X is 0, S, or NRx. In some embodiments, X is 0. In some embodiments, X is S. In some embodiments, X is NRx, wherein Rx is as defined above and described herein.
[0210]x As generally defined above, i R s hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group. In some embodiments, Rx is hydrogen. In some embodiments, Rx is optionally substituted alkyl. In some embodiments, Rx is optionally substituted alkenyl.
In some embodiments, Rx is optionally substituted alkynyl. In some embodiments, Rx is optionally substituted carbocyclyl. In some embodiments, Rx is optionally substituted heterocyclyl. In some embodiments, Rx is optionally substituted aryl. In some embodiments, Rx is optionally substituted heteroaryl. In some embodiments, Rx is a nitrogen protecting group.
[0211] As generally defined above, Y is 0, S, or NR. In some embodiments, Y is 0. In some embodiments, Y is S. In some embodiments, Y is NR, wherein RY is as defined above and described herein.
[0212] As generally defined above, RY is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group. In some embodiments, RY is hydrogen. In some embodiments, RY is optionally substituted alkyl. In some embodiments, RY is optionally substituted alkenyl.
In some embodiments, RY is optionally substituted alkynyl. In some embodiments, RY is is optionally substituted carbocyclyl. In some embodiments, RY is optionally substituted heterocyclyl. In some embodiments, RY is optionally substituted aryl. In some embodiments, RY is is optionally substituted heteroaryl. In some embodiments, RY is a nitrogen protecting group.
[0213] As generally defined above, RP is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom. In some embodiments, RP is hydrogen. In some embodiments, RP is optionally substituted alkyl. In some embodiments, RP is optionally substituted alkenyl. In some embodiments, RP is optionally substituted alkynyl. In some embodiments, RP is optionally substituted carbocyclyl. In some embodiments, RP is optionally substituted heterocyclyl. In some embodiments, RP is optionally substituted aryl. In some embodiments, RP is optionally substituted heteroaryl. In some embodiments, RP is an oxygen protecting group when attached to an oxygen atom.
In some embodiments, RP is a sulfur protecting group when attached to a sulfur atom.
In some embodiments, RP is a nitrogen protecting group when attached to a nitrogen atom.
[0214] As generally defined above, RL is optionally substituted C1_50 alkyl, optionally substituted C2_50 alkenyl, optionally substituted C2_50 alkynyl, optionally substituted heteroCi_so alkyl, optionally substituted heteroC2_50 alkenyl, optionally substituted heteroC2_50 alkynyl, or a polymer.
[0215] In some embodiments, RL is optionally substituted C1_50 alkyl. In some embodiments, RL is optionally substituted C2_50 alkyl. In some embodiments, RL
is optionally substituted C2_40 alkyl. In some embodiments, RL is optionally substituted C2_30 alkyl. In some embodiments, RL is optionally substituted C2_20 alkyl. In some embodiments, RL
is optionally substituted C2_19 alkyl. In some embodiments, RL is optionally substituted C2_18 alkyl. In some embodiments, RL is optionally substituted C2_17 alkyl. In some embodiments, RL
is optionally substituted C2_16 alkyl. In some embodiments, RL is optionally substituted C2_15 alkyl. In some embodiments, RL is optionally substituted C2_14 alkyl. In some embodiments, RL
is optionally substituted C2_13 alkyl. In some embodiments, RL is optionally substituted C2_12 alkyl. In some embodiments, RL is optionally substituted C2_11 alkyl. In some embodiments, RL
is optionally substituted C2_10 alkyl. In some embodiments, RL is optionally substituted C2_9 alkyl. In some embodiments, RL is optionally substituted C2_8 alkyl. In some embodiments, RL
is optionally substituted C2_7 alkyl. In some embodiments, RL is optionally substituted C2_6 alkyl.
[0216] In some embodiments, RL is optionally substituted C4_50 alkyl. In some embodiments, RL is optionally substituted C4_40 alkyl. In some embodiments, RL
is optionally substituted C4_30 alkyl. In some embodiments, RL is optionally substituted C4_20 alkyl. In some embodiments, RL is optionally substituted C4_19 alkyl. In some embodiments, RL
is optionally substituted C4_18 alkyl. In some embodiments, RL is optionally substituted C4_17 alkyl. In some embodiments, RL is optionally substituted C4_16 alkyl. In some embodiments, RL
is optionally substituted C4_15 alkyl. In some embodiments, RL is optionally substituted C4_14 alkyl. In some embodiments, RL is optionally substituted C4_13 alkyl. In some embodiments, RL
is optionally substituted C4_12 alkyl. In some embodiments, RL is optionally substituted C4_11 alkyl. In some embodiments, RL is optionally substituted C4_10 alkyl. In some embodiments, RL
is optionally substituted C4_9 alkyl. In some embodiments, RL is optionally substituted C4_8 alkyl. In some mos ui lic)HE LI-6D polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui .pc)HE
81-6D polmpscins icipuopdo ST _Ii `sluoulTpoqulo mos ui .pc)HE 61-6D polmpscins iCupuopdo ST
_I-N `siuoulTpoqulo mos ui .pc)HE oz-6D polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui .pc)HE
0E-6D polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui liC)HE ot-6D polmpscins iCupuopdo ST
_I-N `siuoulTpoqulo mos ui .pc)HE os-6D pompscins icipuopdo ST _I-N `siuoulTpoqulo mos ui 16IZ0]
lic)HE 6-8D polmpscins icipuopdo ST _Ii `siuoulTpoqulo mos ui .pc)HE 01-8D polmpscins iCupuopdo ST
_I-N `siuoulTpoqulo mos ui liC)HE 11-8D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liC)HE
zI-8D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liC)HE EI-8D polmpsqns iCupuopdo ST
_Ii `siuoulTpoqulo mos ui liC)HE tI-8D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liC)HE
c1-8D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liC)HE 91-8D polmpsqns iCupuopdo ST
_Ii `siuoulTpoqulo mos ui lic)HE LTD polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui .pc)HE
81-8D polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui lic)HE 61-8D polmpscins iCupuopdo ST
_I-N `siuoulTpoqulo mos ui .pc)HE oz-8D polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui .pc)HE
0E-8D polmpscins icipuopdo ST _Ii `siuoulTpoqulo mos ui .pc)HE ot-8D polmpscins iCupuopdo ST
_I-N `siuoulTpoqulo mos ui .pc)HE os-8D pompscins icipuopdo ST _I-N `siuoulTpoqulo mos ui 18IZ0]
lic)HE L-9 D polmpscins iCupuopdo ST _I-N `siuoulTpoqulo mos ui .pc)HE 8-9D pompscins iCupuopdo ST _I-N `siuoulTpoqulo mos ui .pc)HE 6-9D polmpscins icipuopdo ST _Ii `siuoulTpoqulo mos ui .pc)HE 0T-9D polmpscins iCupuopdo ST
_I-N `siuoulTpoqulo mos ui liC)HE 11-9D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liC)HE
z1-9D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liC)HE 1-9D polmpsqns iCupuopdo ST
_Ii `siuoulTpoqulo mos ui liC)HE tI-9D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liC)HE
c1-9D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liC)HE 91-9D polmpsqns iCupuopdo ST
_Ii `siuoulTpoqulo mos ui .pc)HE LI-9D polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui .pc)HE
8T-9D polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui lic)HE 61-9D polmpscins iCupuopdo ST
_I-N `siuoulTpoqulo mos ui .pc)HE oz-9D polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui .pc)HE
0E-9D polmpscins icipuopdo ST ,_11 `siuoulTpoqulo mos ui .pc)HE ot-9D polmpscins iCupuopdo ST
_I-N `siuoulTpoqulo mos ui .pc)HE os-9D pompscins icipuopdo ST _I-N `siuoulTpoqulo mos ui ILIZO]
.pc)HE 9-1,3 polmpsqns ictpuopdo ST _Ii `siuoulTpoqulo mos ui .pc)HE L-17 D polmpscins iCupuopdo ST
_I-N `siuoulTpoqulo 6LI90/tIOZSI1IIDd L9tI90/SIOZ OM

OL
iCHEuopdo ST 1 `siuoulTpoqulo mos ui liC)HE 9i-zip polmpsqns iCupuopdo ST 1 `siuoulTpoqulo mos ui =pc)HE Li-zip palmpscins iCipuopdo ST ,1-N `siuoulTpoqulo mos ui =pc)HE si-ziD polmpscins iCupuopdo ST ,1-N `siuoulTpoqulo mos ui =pc)HE 6I-ZID pOTTITITSCIttS
JCIIPII0pd0 ST ,III `siuoulTpoqulo amos ui =pc)HE ' I)palmpscins iCipuopdo ST ,1-N `siuoulTpoqulo amos ui =pc)HE
' I) polmpscins ictpuopdo ST ,1-N `siuoulTpoqulo mos ui =pc)HE ot-zID palmpscins iCupuopdo ST
,1-N `siuoulTpoqulo mos ui =pc)HE os-zID pompscins icipuopdo ST ,I-N `siuoulTpoqulo mos ui IZZZO]
.pc)HE ZI-HD polmpsqns iCupuopdo ST 1 `siuoulTpoqulo mos ui liC)HE ET-TID polmpsqns iCupuopdo ST 1 `siuoulTpoqulo mos ui .pC)HE ti-IID polmpsqns iCipuopdo ST ,_11 `siuoulTpoqulo mos ui .pC)HE
ci-IID polmpsqns iCupuopdo ST 1 `siuoulTpoqulo mos ui liC)HE 9I-HD polmpsqns iCupuopdo ST 1 `siuoulTpoqulo amos ui =pc)HE Li-TID polmpscins iCipuopdo ST ,1-N `siuoulTpoqulo mos ui =pc)HE 8T-HD palmpscins iCupuopdo ST ,1-N `siuoulTpoqulo mos ui =pc)HE 6I-IID pOTTITITSCIttS
JCIIPII0pd0 ST III `siuoulTpoqulo mos ui =pc)HE oz-HD palmpscins iCipuopdo ST ,1-N `siuoulTpoqulo mos ui =pc)HE
0-11D polmpscins ictpuopdo ST ,1-N `siuoulTpoqulo mos ui =pc)HE ot-HD palmpscins iCupuopdo ST
,1-N `siuoulTpoqulo mos ui =pc)HE os-iiD pompscins icumoRdo ST ,III `siuoulTpoqulo mos ui IIZZO]
=p c)E zc)HE " 'D polmpscins iCipuopdo ST ,I-N `siuoulTpoqulo mos ui =pH I-oID
palmpscins ictpuopdo ST ,1-N `siuoulTpoqulo mos ui =pc)HE Ei-oiD palmpscins iCupuopdo ST
,1-N `siuoulTpoqulo mos ui =pc)HE ti-oiD palmpscins iCipuopdo ST ,1-N `siuoulTpoqulo mos ui =pc)HE si-oiD polmpscins ictpuopdo ST ,1-N `siuoulTpoqulo mos ui =pc)HE 91-01D palmpscins iCupuopdo ST
,1-N `siuoulTpoqulo mos ui =pc)HE Li-oiD polmpscins iCipuopdo ST ,1-N `siuoulTpoqulo mos ui liC)HE si-oiD polmpscins ictpuopdo ST ,1-N `siuoulTpoqulo mos ui =pc)HE 61-01D palmpscins iCupuopdo ST
,1-N `siuoulTpoqulo mos ui =pc)HE oz-oiD palmpscins iCipuopdo ST ,1-N `siuoulTpoqulo mos ui =pc)HE 0-OID polmpscins ictpuopdo ST ,1-N `siuoulTpoqulo mos ui =pc)HE ot-oID palmpscins iCupuopdo ST
,1-N `siuoulTpoqulo mos ui =pc)HE os-oiD pompscins iCipuopdo ST 1-N `siuoulTpoqulo mos ui RIZZO]
lic)HE 0T-6D polmpscins iCupuopdo ST ,1-N `siuoulTpoqulo mos ui .pc)fp H-6D polmpscins iCipuopdo ST 1-N `siuoulTpoqulo mos ui .pc)HE
zI-6D polmpscins icipuopdo ST ,_11 `siuoulTpoqulo mos ui .pc)HE ET-6D polmpscins iCupuopdo ST
,1-N `siuoulTpoqulo mos ui liC)HE 171-6D polmpscins iCipuopdo ST 1-N `siuoulTpoqulo mos ui .pC)HE
c I-6D polmpsqns icipuopdo ST ,_11 `siuoulTpoqulo mos ui .pc)HE 91-6D polmpscins iCupuopdo ST
,1-N `siuoulTpoqulo 6LI90/tIOZSI1IIDd L9tI90/SIOZ OM

substituted C12-15 alkyl. In some embodiments, RL is optionally substituted C12-14 alkyl. In some embodiments, RL is optionally substituted C12-13 alkyl.
[0223] In some embodiments, RL is optionally substituted C6 alkyl. In some embodiments, RL is optionally substituted C7 alkyl. In some embodiments, RL is optionally substituted C8 alkyl. In some embodiments, RL is optionally substituted C9 alkyl. In some embodiments, RL is optionally substituted C10 alkyl. In some embodiments, RL
is optionally substituted Cii alkyl. In some embodiments, RL is optionally substituted C12 alkyl. In some embodiments, RL is optionally substituted C13 alkyl. In some embodiments, RL
is optionally substituted C14 alkyl. In some embodiments, RL is optionally substituted C15 alkyl. In some embodiments, RL is optionally substituted C16 alkyl. In some embodiments, RL
is optionally substituted C17 alkyl. In some embodiments, RL is optionally substituted C18 alkyl. In some embodiments, RL is optionally substituted C19 alkyl. In some embodiments, RL
is optionally substituted C20 alkyl.
[0224] In some embodiments, for example, in any of the above embodiments, RL is a substituted alkyl group. In some embodiments, RL is an unsubstituted alkyl group. In some embodiments, RL is an optionally substituted straight-chain alkyl group. In some embodiments, RL is a substituted straight-chain alkyl group. In some embodiments, RL is an unsubstituted straight-chain alkyl group. In some embodiments, RL is an optionally substituted branched alkyl group. In some embodiments, RL is a substituted branched alkyl group. In some embodiments, RL is an unsubstituted branched alkyl group.
[0225] In certain embodiments, at least one instance of RL is an unsubstituted alkyl.
Exemplary unsubstituted alkyl groups include, but are not limited to, ¨CH3, ¨C2H55 ¨C31475 ¨
C4H9, ¨05H11, ¨C61113, ¨C7H15, ¨C81117, ¨C91119, ¨C101421, ¨C11H23, ¨C1214255 ¨C1314275 ¨C1414295 ¨
C15H31, ¨C16H33, ¨C17H35, ¨C18H37, ¨C19H39, ¨C20H41, ¨C21H43, ¨C22H45, ¨C23H47, ¨C24H49, and -C251451.
[0226] In certain embodiments, at least one instance of RL is a substituted alkyl. For example, in certain embodimenets, at least one instance of RL is an alkyl substituted with one or more fluorine substituents. Exemplary fluorinated alkyl groups include, but are not limited to:

F. F
EFF 1.:;r FF FF F
F F F F X
F 101LAIX)c>(X
F [7 F
FÃ''FFFF F' FF FF FF F
F./ >4 FF FFF FF FF FF F
F F F \ ' ' F . )4)(\CXY-'2( F F Fk FF /
F, \ ' FF . F r 44 t E/FFFFF F, F F FFFF FF FF FF \F
F
FFFFF ,...--..,...
.,.., , . /
\I F, A -__ F'F FFEFFFFFF--F
FFFFFF FF F F FF FF FF F
FF ..--F F F F .
E
'Nry-NA,- V)(\(-7icV(AX-i ) (x) F FFF¨F -FFFFFFFFF
FFFFFFFF F F EFF FFFF FFFF FF F
F Xic," iXx v ---',--- e ic-,--: F FF F F F t, , ),\_ tr F-"Lx:y FF FFFFFF F FF 1.--F -F -F /
F F. F F. F : f F. r: , F,7FF,F FF,FF FF FF FF, F
F=:4,,--,,A yeX V ,AX .....K_ F FFFFFFFF ,s , i i FrrFFFFIFFFFFF
.,õõAõ,.\4..õ,x ,e+\Kice .',...../
F F F F
F F F FF. F/F F F F FF FF Fl- F
FF F cAl \ ' j \\J
F FFFFFFNT r"L-N-.- ---A--- --A-Y.-N-..- 4 NNicA ' FFFIFFE FFFFFFF FFFFF
\ I cs-FFFFFFFFFFF ¨
EFf FifFir F F FFF, FF F F,FFc.\ IF
FE,F F ,F F /F F F FµxF- F....sr F F FiF FtF Ff- F' 17 Fi F F' F F F F F
µ ,-, -F FF r FF F r FF F
[0227] In some embodiments, RL is optionally substituted C2_50 alkenyl. In some embodiments, RL is optionally substituted C2_40 alkenyl. In some embodiments, RL is optionally substituted C2_30 alkenyl. In some embodiments, RL is optionally substituted C2_20 alkenyl. In some embodiments, RL is optionally substituted C2_19 alkenyl. In some embodiments, RL is optionally substituted C2_18 alkenyl. In some embodiments, RL is optionally substituted C2-17 alkenyl. In some embodiments, RL is optionally substituted C2_16 alkenyl. In some embodiments, RL is optionally substituted C2_15 alkenyl. In some embodiments, RL is optionally EL
`siuoulTpoqulo mos ui liCuo3HE 6-9D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liCuo3HE
o1-9D pompsqns iCipuopdo ST _I-N `siuoulTpoqulo aims ui licuo3HE I1-9D
polmpscins iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuo3HE z1-9D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos uI .1,Cuo3IIP 1-9D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liCu03up tI-9D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuo3up cI-9D polmpsqns iCupuopdo ST
_Ii `siuoulTpoqulo mos ui liCu03up 91-9D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liCu03up LI-9D pompscins iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1Jcuo3up 8 1-9D
polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui 1Jcuo3up 61-9D polmpscins iCipuopdo ST _I-N
`siuoulTpoqulo mos uI *IiCuo3IIP oz-6D polmpscins iCipuopdo ST _Ii `siuoulTpoqulo mos ui .pcu03up 9E-9D polmpscins icipuopdo ST _I-N `siuoulTpoqulo mos ui 1icuo3up 9t-9D polmpscins iCupuopdo ST _I-N `siuoulTpoqulo mos ui .pcu03up 0c-9D pompscins icipuopdo ST _I-N `siuoulTpoqulo mos ui 16ZZ0]
1iCuo3up 9-17D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuo3up L-tp polmpscins iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1Jcuo3up 8-17D
polmpscins iCipuopdo sT _I-N
`siuoulTpoqulo mos ui 1iCuo3up 6-tp polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuo3up OT-tp pompsqns iCipuopdo ST _I-N `siuoulTpoqulo mos ui 1icuo3up "D polmpscins iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuo3up zI-17D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos uI *IiCuo3IIP EI-tD polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui 11Cu03up 171-17D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuo3up cI-17D polmpsqns iCupuopdo ST
_Ii `siuoulTpoqulo mos ui 1iCuo3up 9i-17D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuo3up LTD pompscins iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1Jcuo3up 8 T-tp polmpscins iCipuopdo STTI-N `siuoulTpoqulo mos ui 1Jcuo3up 6i-17D polmpscins iCipuopdo ST _I-N
`siuoulTpoqulo mos uI *IiCuo3IIP oz-tp polmpscins iCipuopdo ST _Ii `siuoulTpoqulo mos ui .pcu03up 9E-to polmpscins icipuopdo ST _I-N `siuoulTpoqulo mos ui 1icuo3up 0t-tD polmpscins iCupuopdo ST _I-N `siuoulTpoqulo mos ui 1Jcuo3up 9c-17D pompscins icipuopdo ST _I-N `siuoulTpoqulo mos ui 18ZZ0]
1iCuo3up 9-zD polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuo3up L-zp polmpscins iCipuopdo ST ,_11 `siuoulTpoqulo mos ui 1Jcuo3up 8-Z3 polmpscins iCipuopdo `siuoulTpoqulo mos ui 1iCuo3up 6-zD polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuo3up oi-zp pompsqns iCipuopdo ST _I-N `siuoulTpoqulo mos ui 1icuo3up I T-zD
polmpscins iCipuopdo ST ,_11 `siuoulTpoqulo mos ui 1iCuo3up D
polmpsqns iCipuopdo ST `siuoulTpoqulo mos uI *IiCuo3IIP EI-zD polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liCu03up 17I-zD polmpsqns 6LI90/tIOZSI1IIDd L9tI90/SIOZ OM

t L
mos ui lic1103flp 9I-OI D palmpscins iCipuopdo ST _I-N `siuoulTpoqulo alms ui liCuo3HE
Li-oiD palmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui .pcuo3HE si-oiD
palmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui 1icuo3HE 61-0iD palmpscins iCipuopdo ST _I-N
`siuoulTpoqulo mos uI *IiCu33IIB oz-oiD polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui Itc1103Hp 0-OI D palmpscins icipuopdo ST _I-N `siuoulTpoqulo mos ui 1icuo3up ot-oiD palmpscins iCupuopdo ST _I-N `siuoulTpoqulo mos ui licuo3up os-oID pompscins icipuopdo ST _I-N `siuoulTpoqulo mos ui IMO]
.pcuo3up o1-6D pompsqns iCipuopdo ST _I-N `siuoulTpoqulo mos ui licuo3up 11-6D
polmpscins iCipuopdo ST _I-N `siuoulTpoqulo alms ui 1Jcuo3up zI-6D polmpscins iCipuopdo ST _I-N
`siuoulTpoqulo alms uI *IiCuo3IIP E1-6D polmpscins iCipuopdo ST _Ii `siuoulTpoqulo alms ui .pcuo3up 1i-6D polmpscins icipuopdo ST _I-N `siuoulTpoqulo alms ui 1icuo3up si-6D polmpscins iCupuopdo ST _I-N `siuoulTpoqulo alms ui liCuo3up 91-6D polmpscins iCipuopdo ST _I-N `siuoulTpoqulo alms ui liCuo3up LI-6D pompsqns iCipuopdo ST _I-N `siuoulTpoqulo alms ui 1icuo3up ST-6D
polmpscins iCipuopdo ST _I-N `siuoulTpoqulo alms ui 1Jcuo3up 61-6D polmpscins iCipuopdo ST _I-N
`siuoulTpoqulo alms uI *IiCuo3IIP oz-6D polmpscins iCipuopdo ST _Ii `siuoulTpoqulo alms ui .pcu03up 0E-6D polmpscins icipuopdo ST _I-N `siuoulTpoqulo alms ui 1icuo3up 0t-6D polmpscins iCupuopdo ST _I-N `siuoulTpoqulo alms ui 1iCuo3up 9c-6D pompscins iCipuopdo ST _I-N `siuoulTpoqulo alms ui ITEZO]
1icuo3up 6-8D polmpscins iCipuopdo ST _Ii `siuoulTpoqulo alms ui 1iCuo3up OT-sp pompsqns iCipuopdo ST _I-N `siuoulTpoqulo alms ui 1icuo3up H-8D
polmpscins iCipuopdo ST _Ii `siuoulTpoqulo alms ui 1iCuo3up z1-8D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo alms uI *IiCuo3IIP EI-8D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo alms ui 11Cu03up tI-8D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo alms ui 1iCuo3up c1-8D polmpsqns iCupuopdo ST
_Ii `siuoulTpoqulo alms ui 1iCuo3up 91-8D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo alms ui 1iCuo3up LI-sp pompscins iCipuopdo ST _Ii `siuoulTpoqulo alms ui 1Jcuo3up 81-8D
polmpscins iCipuopdo ST _I-N `siuoulTpoqulo alms ui 1icuo3up 61-8D polmpscins iCipuopdo ST _I-N
`siuoulTpoqulo alms uI *IiCuo3IIP oz-sp polmpscins iCipuopdo ST _Ii `siuoulTpoqulo alms ui .pcu03up 0E-8D polmpscins icipuopdo ST _I-N `siuoulTpoqulo alms ui 1icuo3up 0i7-8D polmpscins iCupuopdo ST _I-N `siuoulTpoqulo alms ui 1Jcuo3up 9c-8D pompscins icipuopdo STTI-N `siuoulTpoqulo alms ui 10Z0]
.pcuo3up L-9D polmpscins iCipuopdo ST _Ii `siuoulTpoqulo alms ui 1Jcuo3up 8-9D
polmpscins iCipuopdo sT _I-N
6LI90/tIOZSI1IIDd L9tI90/SIOZ OM

embodiments, RL is optionally substituted C10-15 alkenyl. In some embodiments, RL is optionally substituted C10_14 alkenyl. In some embodiments, RL is optionally substituted C10_13 alkenyl. In some embodiments, RL is optionally substituted Cm-12 alkenyl. In some embodiments, RL is optionally substituted Cio-ii alkenyl.
[0233] In some embodiments, RL is optionally substituted C11-50 alkenyl.
In some embodiments, RL is optionally substituted C11-40 alkenyl. In some embodiments, RL is optionally substituted C11_30 alkenyl. In some embodiments, RL is optionally substituted Cil_20 alkenyl. In some embodiments, RL is optionally substituted C11-19 alkenyl. In some embodiments, RL is optionally substituted C11_18 alkenyl. In some embodiments, RL is optionally substituted C11_17 alkenyl. In some embodiments, RL is optionally substituted C11-16 alkenyl. In some embodiments, RL is optionally substituted C11-15 alkenyl. In some embodiments, RL is optionally substituted C11_14 alkenyl. In some embodiments, RL is optionally substituted C11_13 alkenyl. In some embodiments, RL is optionally substituted C11-12 alkenyl.
[0234] In some embodiments, RL is optionally substituted C12-50 alkenyl.
In some embodiments, RL is optionally substituted C12-40 alkenyl. In some embodiments, RL is optionally substituted C12_30 alkenyl. In some embodiments, RL is optionally substituted C12_20 alkenyl. In some embodiments, RL is optionally substituted C12-19 alkenyl. In some embodiments, RL is optionally substituted C12_18 alkenyl. In some embodiments, RL is optionally substituted C12_17 alkenyl. In some embodiments, RL is optionally substituted C12-16 alkenyl. In some embodiments, RL is optionally substituted C12-15 alkenyl. In some embodiments, RL is optionally substituted C12_14 alkenyl. In some embodiments, RL is optionally substituted C12_13 alkenyl.
[0235] In some embodiments, RL is optionally substituted C6 alkenyl. In some embodiments, RL is optionally substituted C7 alkenyl. In some embodiments, RL
is optionally substituted C8 alkenyl. In some embodiments, RL is optionally substituted C9 alkenyl. In some embodiments, RL is optionally substituted Cio alkenyl. In some embodiments, RL
is optionally substituted C11 alkenyl. In some embodiments, RL is optionally substituted Ci2 alkenyl. In some embodiments, RL is optionally substituted C13 alkenyl. In some embodiments, RL
is optionally substituted C14 alkenyl. In some embodiments, RL is optionally substituted C15 alkenyl. In some embodiments, RL is optionally substituted Ci6 alkenyl. In some embodiments, RL
is optionally substituted C17 alkenyl. In some embodiments, RL is optionally substituted C18 alkenyl. In some embodiments, RL is optionally substituted C10 alkenyl. In some embodiments, RL
is optionally substituted C20 alkenyl.
[0236] In some embodiments, for example, in any of the above embodiments, RL is a substituted alkyl group. In some embodiments, RL is an unsubstituted alkyl group. In some embodiments, RL is an optionally substituted straight-chain alkenyl group. In some embodiments, RL is a substituted straight-chain alkenyl group. In some embodiments, RL is an unsubstituted straight-chain alkenyl group. In some embodiments, RL is an optionally substituted branched alkenyl group. In some embodiments, RL is a substituted branched alkenyl group. In some embodiments, RL is an unsubstituted branched alkenyl group.
[0237] Exemplary unsubstituted alkenyl group include, but are not limited to:
rr 4"

Mytistoleic .4CH.2.),7(71-1.CH(Cf12)3043, Palinitolie.c -(CH2)7CFF-----CH(CH1)5CH3, .Sapienic -(CHa),}CE-1=C11(CII2)CH3, Oleic --(012)7CII=CI1(012)7CH:3, Linoteic -(CH2)7CIF-CHCI-1201,CH(C1-12)4CH3µ
(x.-Linolenic -(CF12)7CH=CEICH2,01-=0-K7H201=C.FICH2CI3, Arachinodonie -(CH2):CH=CHCH2CF1.---CHCH=CH=CFICH2CH=C:171(CH2).4CH3, Eicosaperitnenoic -(CF1.1),3CII=CHCH2.0-1=0-K7H2CI-1.CHCRICH.CHCF12.01,,CHC112.CIT3, Erode -(CH) CH=CH(CH2)7CF4, and Docosahexaenai -c (C1-12)2CHHCHCHS1-1.--ClICH2CH.CHCH2C.H.CHCHAII.(71-1CH20-1,-C
H-CHaCHs [0238] In some embodiments, wherein RL is defined as a C6_50a1ky1 or C6_50alkenyl groups, such groups are meant to encompass lipophilic groups (also referred to as a "lipid tail").
Lipophilic groups comprise a group of molecules that include fats, waxes, oils, fatty acids, and the like. Lipid tails present in these lipid groups can be saturated and unsaturated, depending on whether or not the lipid tail comprises double bonds. The lipid tail can also comprise different lengths, often categorized as medium (i.e., with tails between 7-12 carbons, e.g., C7_12 alkyl or C7_12 alkenyl), long (i.e., with tails greater than 12 carbons and up to 22 carbons, e.g., C13-22a1ky1 or C13-22 alkenyl), or very long (i.e., with tails greater than 22 carbons, e.g., C23-30 alkyl or C23-30 alkenyl).
[0239] In some embodiments, RL is optionally substituted C2_50 alkynyl.
In some embodiments, RL is optionally substituted C2_40 alkynyl. In some embodiments, RL is optionally substituted C2_30 alkynyl. In some embodiments, RL is optionally substituted C2_20 alkynyl. In some embodiments, RL is optionally substituted C2_19 alkynyl. In some embodiments, RL is optionally substituted C2_18 alkynyl. In some embodiments, RL is optionally substituted C2_17 alkynyl. In some embodiments, RL is optionally substituted C2_16 alkynyl. In some embodiments, RL is optionally substituted C2_15 alkynyl. In some embodiments, RL is optionally substituted C2_14 alkynyl. In some embodiments, RL is optionally substituted C2_13 alkynyl. In some embodiments, RL is optionally substituted C2_12 alkynyl. In some embodiments, RL is *pcuiC3HE
L-9D polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui 11Cu1c3HE 8-9D
polmpscins iCipuopdo ST _I-N
`siuoulTpoqulo mos ui 11Cu1C3HE 6-9D polmpsqns iCipuopdo sT _Ii `siuoulTpoqulo mos ui 11Cu1C3HE
o1-6D polmpscins iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuiC3HE 11-9D
polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuiC3HE z1-9D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos uI *IiCuiC3IIP 1-9D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liCu1C3up tI-9D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuiC3up cI-9D polmpsqns iCupuopdo ST
_Ii `siuoulTpoqulo mos ui liCu1C3up 91-9D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liCu1C3up LI-6D polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui liCuic3up 8 1-9D
polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui 1iCuic3up 61-9D polmpscins iCipuopdo ST _I-N
`siuoulTpoqulo mos uI *IiCuiC3IIP oz-6D polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui liCuic3up 0E-9D polmpscins icipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuic3up 017-9D polmpscins iCupuopdo ST _I-N `siuoulTpoqulo mos ui liCu1c3up 0s-9D pompscins iCipuopcIo ST _I-N `siuoulTpoqulo mos ui Iirzol 1iCuiC3up 9-tp polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuiC3up p polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui 1iCuic3up 8-17D
polmpscins iCipuopdo sT _I-N
`siuoulTpoqulo mos ui liCu1C3up 6-tp polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuiC3up cm-fp polmpscins iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuiC3up 11-tD
polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liCu1C3up zI-17D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos uI *IiCu1C3IIP EI-tD polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liCu1C3up 171-17D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuiC3up c I-17D polmpsqns iCupuopdo ST _Ii `siuoulTpoqulo mos ui liCu1C3up 9I-tD polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liCu1C3up LTD polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui 1iCuic3up 8 T-tp polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui liCu1c3up 6i-tD polmpscins iCipuopdo ST _I-N
`siuoulTpoqulo mos uI *IiCu1C3IIP oz-tp polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui liCu1c3up 0E-tD polmpscins icipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuic3up 017-17D polmpscins iCupuopdo ST _I-N `siuoulTpoqulo mos ui liCu1c3up os-tD pompscins icipuopcIo ST _I-N `siuoulTpoqulo mos ui Iorzol .pcu1c3up 9-zp polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuiC3up L-zp polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui 1iCuic3up 8-Z D
polmpscins iCipuopdo sT _I-N
`siuoulTpoqulo mos ui 1iCuiC3up 6-zD polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuiC3up oi-zp polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui 1iCuic3up "D
polmpscins iCipuopdo 6LI90/tIOZSI1IIDd L9tI90/SIOZ OM

ui .PCIIA3HP ET-OID palmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui .pcuic3up ti-oiD polmpscins ictpuopdo ST _I-N `siuoulTpoqulo mos ui 1Jcuic3up sT-oID palmpscins iCupuopdo ST _I-N `siuoulTpoqulo mos ui .pcu1c3up 9i-oiD palmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui liCu1C3up Li-oiD polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui 1icuic3up sT-oID
polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui 1Jcuic3up 61-0iD palmpscins iCipuopdo ST _I-N
`siuoulTpoqulo mos uI *IiCuiC3IIP oz-oiD palmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui .pcu1c3up oE-oiD polmpscins ictpuopdo ST _I-N `siuoulTpoqulo mos ui 1Jcuic3up ot-oiD palmpscins iCupuopdo ST _I-N `siuoulTpoqulo mos ui licli1c3up 05-0ID pompscins iCipuopdo ST _Ii `siuoulTpoqulo mos ui Itrzol .pcuic3up 01-6D polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui liCuic3up H-6D
polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui 1iCuic3up zI-6D polmpscins iCipuopdo ST _I-N
`siuoulTpoqulo mos uI *IiCu1C3IIP E1-6D polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui liCu1c3up tI-6D polmpscins icipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuic3up si-6D polmpscins iCupuopdo ST _I-N `siuoulTpoqulo mos ui liCu1c3up 91-6D polmpscins iCipuopdo ST _Ii `siuoulTpoqulo mos ui liCu1C3up LI-6D polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui liCu1c3up 81-6D
polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui liCu1c3up 61-6D polmpscins iCipuopdo ST _I-N
`siuoulTpoqulo mos uI *IiCu1C3IIP oz-6D polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui liCu1c3up 0E-6D polmpscins icipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuic3up ot-6D polmpscins iCupuopdo ST _I-N `siuoulTpoqulo mos ui liCu1c3up os-6D pompscins icipuopdo ST _I-N `siuoulTpoqulo mos ui [Erzo]
1icuic3up 6-8D polmpscins iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuiC3up OT-sp polmpscins iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuiC3up II-8D
polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liCu1C3up zI-8D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos uI *IiCu1C3IIP ET-8D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liCu1C3up tI-8D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuiC3up cI-8D polmpsqns iCupuopdo ST
_Ii `siuoulTpoqulo mos ui liCu1C3up 91-8D polmpsqns iCipuopdo ST _Ii `siuoulTpoqulo mos ui liCu1C3up LI-sp polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui 1iCuic3up 81-8D
polmpscins iCipuopdo ST _I-N `siuoulTpoqulo mos ui liCu1c3up 61-8D polmpscins iCipuopdo ST _I-N
`siuoulTpoqulo mos uI *IiCuiC3IIP oz-sp polmpscins iCipuopdo ST 1-N `siuoulTpoqulo mos ui 1iCuic3up 0E-8D polmpscins icipuopdo ST _Ii `siuoulTpoqulo mos ui 1iCuic3up 017-8D polmpscins iCupuopdo ST _I-N `siuoulTpoqulo mos ui 1iCuic3up os-sp pompscins icipuopdo ST _I-N `siuoulTpoqulo mos ui Izrzol 6LI90/tIOZSI1IIDd L9tI90/SIOZ OM

some embodiments, RL is optionally substituted Cm-12 alkynyl. In some embodiments, RL is optionally substituted C10_11 alkynyl.
[0245] In some embodiments, RL is optionally substituted C11-50 alkynyl.
In some embodiments, RL is optionally substituted C11-40 alkynyl. In some embodiments, RL is optionally substituted C11-30 alkynyl. In some embodiments, RL is optionally substituted Cil_20 alkynyl. In some embodiments, RL is optionally substituted C11-19 alkynyl. In some embodiments, RL is optionally substituted C11_18 alkynyl. In some embodiments, RL is optionally substituted C11_17 alkynyl. In some embodiments, RL is optionally substituted Cii-i6 alkynyl. In some embodiments, RL is optionally substituted C11-15 alkynyl. In some embodiments, RL is optionally substituted C11_14 alkynyl. In some embodiments, RL is optionally substituted C11_13 alkynyl. In some embodiments, RL is optionally substituted C11-12 alkynyl.
[0246] In some embodiments, RL is optionally substituted C12-50 alkynyl.
In some embodiments, RL is optionally substituted C12-40 alkynyl. In some embodiments, RL is optionally substituted C12_30 alkynyl. In some embodiments, RL is optionally substituted C12_20 alkynyl. In some embodiments, RL is optionally substituted C12-19 alkynyl. In some embodiments, RL is optionally substituted C12_18 alkynyl. In some embodiments, RL is optionally substituted C12_17 alkynyl. In some embodiments, RL is optionally substituted C12_16 alkynyl. In some embodiments, RL is optionally substituted C12-15 alkynyl. In some embodiments, RL is optionally substituted C1214 alkynyl. In some embodiments, RL is optionally substituted C12_13 alkynyl.
[0247] In some embodiments, RL is optionally substituted C6 alkynyl. In some embodiments, RL is optionally substituted C7 alkynyl. In some embodiments, RL
is optionally substituted C8 alkynyl. In some embodiments, RL is optionally substituted C9 alkynyl. In some embodiments, RL is optionally substituted C10 alkynyl. In some embodiments, RL
is optionally substituted C 1 1 alkynyl. In some embodiments, RL is optionally substituted C12 alkynyl. In some embodiments, RL is optionally substituted C13 alkynyl. In some embodiments, RL
is optionally substituted Ci4 alkynyl. In some embodiments, RL is optionally substituted C15 alkynyl. In some embodiments, RL is optionally substituted C16 alkynyl. In some embodiments, RL
is optionally substituted Ci7 alkynyl. In some embodiments, RL is optionally substituted C18 alkynyl. In some embodiments, RL is optionally substituted Ci9 alkynyl. In some embodiments, RL
is optionally substituted C20 alkynyl.

[0248] In some embodiments, for example, in any of the above embodiments, RL is a substituted alkynyl group. In some embodiments, RL is an unsubstituted alkynyl group. In some embodiments, RL is an optionally substituted straight-chain alkyl group. In some embodiments, RL is an optionally substituted straight-chain alkynyl group. In some embodiments, RL is a substituted straight-chain alkynyl group. In some embodiments, RL is an unsubstituted straight-chain alkynyl group. In some embodiments, RL is an optionally substituted branched alkynyl group. In some embodiments, RL is a substituted branched alkynyl group. In some embodiments, RL is an unsubstituted branched alkynyl group.
[0249] In some embodiments, RL is optionally substituted heteroCi_soalkyl. In some embodiments, RL is optionally substituted heteroC2_50alkyl. In some embodiments, RL is optionally substituted heteroC2_40alkyl. In some embodiments, RL is optionally substituted heteroC2_30alkyl. In some embodiments, RL is optionally substituted heteroC2_20alkyl. In some embodiments, RL is optionally substituted heteroC249alkyl. In some embodiments, RL is optionally substituted heteroC248alkyl. In some embodiments, RL is optionally substituted heteroC247alkyl. In some embodiments, RL is optionally substituted heteroC246alkyl. In some embodiments, RL is optionally substituted heteroC245alkyl. In some embodiments, RL is optionally substituted heteroC244alkyl. In some embodiments, RL is optionally substituted heteroC243alkyl. In some embodiments, RL is optionally substituted heteroC242alkyl. In some embodiments, RL is optionally substituted heteroC2_1ialkyl. In some embodiments, RL is optionally substituted heteroC240alkyl. In some embodiments, RL is optionally substituted heteroC2_9alkyl. In some embodiments, RL is optionally substituted heteroC2_8alkyl. In some embodiments, RL is optionally substituted heteroC2_7alkyl. In some embodiments, RL is optionally substituted heteroC2_6alkyl.
[0250] In some embodiments, RL is optionally substituted heteroC4_50alkyl. In some embodiments, RL is optionally substituted heteroC4_40alkyl. In some embodiments, RL is optionally substituted heteroC4_30alkyl. In some embodiments, RL is optionally substituted heteroC4_20alkyl. In some embodiments, RL is optionally substituted heteroC449alkyl. In some embodiments, RL is optionally substituted heteroC448alkyl. In some embodiments, RL is optionally substituted heteroC447alkyl. In some embodiments, RL is optionally substituted heteroC446alkyl. In some embodiments, RL is optionally substituted heteroC445alkyl. In some embodiments, RL is optionally substituted heteroC444alkyl. In some embodiments, RL is optionally substituted heteroC443alkyl. In some embodiments, RL is optionally substituted heteroC442alkyl. In some embodiments, RL is optionally substituted heteroC4_1ialkyl. In some embodiments, RL is optionally substituted heteroC440alkyl. In some embodiments, RL is optionally substituted heteroC4_9alkyl. In some embodiments, RL is optionally substituted heteroC4_8alkyl. In some embodiments, RL is optionally substituted heteroC4_7alkyl. In some embodiments, RL is optionally substituted heteroC4_6alkyl.
[0251] In some embodiments, RL is optionally substituted heteroC6_50alkyl. In some embodiments, RL is optionally substituted heteroC6_40alkyl. In some embodiments, RL is optionally substituted heteroC6_30alkyl. In some embodiments, RL is optionally substituted heteroC6_20alkyl. In some embodiments, RL is optionally substituted heteroC649alkyl. In some embodiments, RL is optionally substituted heteroC648alkyl. In some embodiments, RL is optionally substituted heteroC647alkyl. In some embodiments, RL is optionally substituted heteroC646alkyl. In some embodiments, RL is optionally substituted heteroC645alkyl. In some embodiments, RL is optionally substituted heteroC644alkyl. In some embodiments, RL is optionally substituted heteroC643alkyl. In some embodiments, RL is optionally substituted heteroC642alkyl. In some embodiments, RL is optionally substituted heteroC6_1ialkyl. In some embodiments, RL is optionally substituted heteroC640alkyl. In some embodiments, RL is optionally substituted heteroC6_9alkyl. In some embodiments, RL is optionally substituted heteroC6_8alkyl. In some embodiments, RL is optionally substituted heteroC6_7alkyl.
[0252] In some embodiments, RL is optionally substituted heteroC8_50alkyl. In some embodiments, RL is optionally substituted heteroC8_40alkyl. In some embodiments, RL is optionally substituted heteroC8_30alkyl. In some embodiments, RL is optionally substituted heteroC8_20alkyl. In some embodiments, RL is optionally substituted heteroC849alkyl. In some embodiments, RL is optionally substituted heteroC848alkyl. In some embodiments, RL is optionally substituted heteroC847alkyl. In some embodiments, RL is optionally substituted heteroC846alkyl. In some embodiments, RL is optionally substituted heteroC845alkyl. In some embodiments, RL is optionally substituted heteroC844alkyl. In some embodiments, RL is optionally substituted heteroC843alkyl. In some embodiments, RL is optionally substituted heteroC842alkyl. In some embodiments, RL is optionally substituted heteroC8_1ialkyl. In some embodiments, RL is optionally substituted heteroC8_10alkyl. In some embodiments, RL is optionally substituted heteroC8_9alkyl.
[0253] In some embodiments, RL is optionally substituted heteroC9_50alkyl. In some embodiments, RL is optionally substituted heteroC9_40alkyl. In some embodiments, RL is optionally substituted heteroC9_30alkyl. In some embodiments, RL is optionally substituted heteroC9_20alkyl. In some embodiments, RL is optionally substituted heteroC9_19alkyl. In some embodiments, RL is optionally substituted heteroC9_18alkyl. In some embodiments, RL is optionally substituted heteroC9_17alkyl. In some embodiments, RL is optionally substituted heteroC9_16alkyl. In some embodiments, RL is optionally substituted heteroC9_15alkyl. In some embodiments, RL is optionally substituted heteroC9_14alkyl. In some embodiments, RL is optionally substituted heteroC9_13alkyl. In some embodiments, RL is optionally substituted heteroC9_12alkyl. In some embodiments, RL is optionally substituted heteroC9_1ialkyl. In some embodiments, RL is optionally substituted heteroC9_10alkyl.
[0254] In some embodiments, RL is optionally substituted heteroCio-soalkyl. In some embodiments, RL is optionally substituted heteroCio-4oalkyl. In some embodiments, RL is optionally substituted heteroCio-3oalkyl. In some embodiments, RL is optionally substituted heteroCio_20alkyl. In some embodiments, RL is optionally substituted heteroCio_19alkyl. In some embodiments, RL is optionally substituted heteroCio-isalkyl. In some embodiments, RL is optionally substituted heteroCio-ralkyl. In some embodiments, RL is optionally substituted heteroCio_malkyl. In some embodiments, RL is optionally substituted heteroC10_15alkyl. In some embodiments, RL is optionally substituted heteroCio-malkyl. In some embodiments, RL is optionally substituted heteroCio-Dalkyl. In some embodiments, RL is optionally substituted heteroC io-ualkyl. In some embodiments, RL is optionally substituted heteroCio-iialkyl.
[0255] In some embodiments, RL is optionally substituted heteroCii-soalkyl. In some embodiments, RL is optionally substituted heteroC1i-4oalkyl. In some embodiments, RL is optionally substituted heteroC1i-3oalkyl. In some embodiments, RL is optionally substituted heteroC1i_20alkyl. In some embodiments, RL is optionally substituted heteroC1i_i9alkyl. In some embodiments, RL is optionally substituted heteroCii-isalkyl. In some embodiments, RL is optionally substituted heteroCii-ralkyl. In some embodiments, RL is optionally substituted heteroCii_malkyl. In some embodiments, RL is optionally substituted heteroC1i_i5alkyl. In some embodiments, RL is optionally substituted heteroC11-i4alkyl. In some embodiments, RL is optionally substituted heteroCii-Dalkyl. In some embodiments, RL is optionally substituted hetero C ii-ualkyl.
[0256] In some embodiments, RL is optionally substituted heteroCi2-5oalkyl. In some embodiments, RL is optionally substituted heteroCi2-4oalkyl. In some embodiments, RL is optionally substituted heteroCi2-3oalkyl. In some embodiments, RL is optionally substituted heteroCi2_20alkyl. In some embodiments, RL is optionally substituted heteroCi2_19alkyl. In some embodiments, RL is optionally substituted heteroCi2-18alkyl. In some embodiments, RL is optionally substituted heteroCi2-ralkyl. In some embodiments, RL is optionally substituted heteroCi2_16alkyl. In some embodiments, RL is optionally substituted heteroC12_15alkyl. In some embodiments, RL is optionally substituted heteroCi2-14alkyl. In some embodiments, RL is optionally substituted heteroC12-13alkyl.
[0257] In some embodiments, RL is optionally substituted heteroC6alkyl.
In some embodiments, RL is optionally substituted heteroC7alkyl. In some embodiments, RL is optionally substituted heteroCsalkyl. In some embodiments, RL is optionally substituted heteroC9alkyl. In some embodiments, RL is optionally substituted heteroCi0alkyl. In some embodiments, RL is optionally substituted heteroCiialkyl. In some embodiments, RL is optionally substituted heteroC ualkyl. In some embodiments, RL is optionally substituted heteroC
nalkyl. In some embodiments, RL is optionally substituted heteroCi4alkyl. In some embodiments, RL is optionally substituted heteroCi5alkyl. In some embodiments, RL is optionally substituted heteroCi6alkyl. In some embodiments, RL is optionally substituted heteroC
ralkyl. In some embodiments, RL is optionally substituted heteroCi8alkyl. In some embodiments, RL is optionally substituted heteroCi9alkyl. In some embodiments, RL is optionally substituted heteroC2oalkyl.
[0258] In some embodiments, for example, in any of the above embodiments, RL is a substituted heteroalkyl group. In some embodiments, RL is an unsubstituted heteroalkyl group.
In some embodiments, RL is an optionally substituted straight-chain heteroalkyl group. In some embodiments, RL is a substituted straight-chain heteroalkyl group. In some embodiments, RL is an unsubstituted straight-chain heteroalkyl group. In some embodiments, RL is an optionally substituted branched heteroalkyl group. In some embodiments, RL is a substituted branched heteroalkyl group. In some embodiments, RL is an unsubstituted branched heteroalkyl group.
[0259] Exemplary unsubstituted heteroalkyl groups include, but are not limited to:
o OF"V
0 ,re cre"'4,;04.
[0260] In some embodiments, RL is optionally substituted heteroC2_50alkenyl. In some embodiments, RL is optionally substituted heteroC2_40alkenyl. In some embodiments, RL is optionally substituted heteroC2_30alkenyl. In some embodiments, RL is optionally substituted heteroC2_20alkenyl. In some embodiments, RL is optionally substituted heteroC249alkenyl. In some embodiments, RL is optionally substituted heteroC248alkenyl. In some embodiments, RL is optionally substituted heteroC247alkenyl. In some embodiments, RL is optionally substituted heteroC246alkenyl. In some embodiments, RL is optionally substituted heteroC245alkenyl. In some embodiments, RL is optionally substituted heteroC2_14alkenyl. In some embodiments, RL is optionally substituted heteroC243alkenyl. In some embodiments, RL is optionally substituted heteroC242alkenyl. In some embodiments, RL is optionally substituted heteroC2_1ialkenyl. In some embodiments, RL is optionally substituted heteroC240alkenyl. In some embodiments, RL is optionally substituted heteroC2_9alkenyl. In some embodiments, RL is optionally substituted heteroC2_8alkenyl. In some embodiments, RL is optionally substituted heteroC2_7alkenyl. In some embodiments, RL is optionally substituted heteroC2_6alkenyl.
[0261] In some embodiments, RL is optionally substituted heteroC4_50alkenyl. In some embodiments, RL is optionally substituted heteroC4_40alkenyl. In some embodiments, RL is optionally substituted heteroC4_30alkenyl. In some embodiments, RL is optionally substituted heteroC4_20alkenyl. In some embodiments, RL is optionally substituted heteroC449alkenyl. In some embodiments, RL is optionally substituted heteroC448alkenyl. In some embodiments, RL is optionally substituted heteroC447alkenyl. In some embodiments, RL is optionally substituted heteroC446alkenyl. In some embodiments, RL is optionally substituted heteroC445alkenyl. In some embodiments, RL is optionally substituted heteroC444alkenyl. In some embodiments, RL is optionally substituted heteroC443alkenyl. In some embodiments, RL is optionally substituted heteroC442alkenyl. In some embodiments, RL is optionally substituted heteroC4_1ialkenyl. In some embodiments, RL is optionally substituted heteroC440alkenyl. In some embodiments, RL is optionally substituted heteroC4_9alkenyl. In some embodiments, RL is optionally substituted heteroC4_8alkenyl. In some embodiments, RL is optionally substituted heteroC4_7alkenyl. In some embodiments, RL is optionally substituted heteroC4_6alkenyl.
[0262] In some embodiments, RL is optionally substituted heteroC6_50alkenyl. In some embodiments, RL is optionally substituted heteroC6_40alkenyl. In some embodiments, RL is optionally substituted heteroC6_30alkenyl. In some embodiments, RL is optionally substituted heteroC6_20alkenyl. In some embodiments, RL is optionally substituted heteroC649alkenyl. In some embodiments, RL is optionally substituted heteroC648alkenyl. In some embodiments, RL is optionally substituted heteroC647alkenyl. In some embodiments, RL is optionally substituted heteroC646alkenyl. In some embodiments, RL is optionally substituted heteroC645alkenyl. In some embodiments, RL is optionally substituted heteroC644alkenyl. In some embodiments, RL is optionally substituted heteroC643alkenyl. In some embodiments, RL is optionally substituted heteroC642alkenyl. In some embodiments, RL is optionally substituted heteroC6_1ialkenyl. In some embodiments, RL is optionally substituted heteroC640alkenyl. In some embodiments, RL is optionally substituted heteroC6_9alkenyl. In some embodiments, RL is optionally substituted heteroC6_8alkenyl. In some embodiments, RL is optionally substituted heteroC6_7alkenyl.

[0263] In some embodiments, RL is optionally substituted heteroC8_50alkenyl. In some embodiments, RL is optionally substituted heteroC8_40alkenyl. In some embodiments, RL is optionally substituted heteroC8_30alkenyl. In some embodiments, RL is optionally substituted heteroC8_20alkenyl. In some embodiments, RL is optionally substituted heteroC849alkenyl. In some embodiments, RL is optionally substituted heteroC848alkenyl. In some embodiments, RL is optionally substituted heteroC8_17alkenyl. In some embodiments, RL is optionally substituted heteroC8-16alkenyl. In some embodiments, RL is optionally substituted heteroC8_15alkenyl. In some embodiments, RL is optionally substituted heteroC844alkenyl. In some embodiments, RL is optionally substituted heteroC843alkenyl. In some embodiments, RL is optionally substituted heteroC842alkenyl. In some embodiments, RL is optionally substituted heteroC8_1ialkenyl. In some embodiments, RL is optionally substituted heteroC840alkenyl. In some embodiments, RL is optionally substituted heteroC8_9alkenyl.
[0264] In some embodiments, RL is optionally substituted heteroC9_50alkenyl. In some embodiments, RL is optionally substituted heteroC9_40alkenyl. In some embodiments, RL is optionally substituted heteroC9_30alkenyl. In some embodiments, RL is optionally substituted heteroC9_20alkenyl. In some embodiments, RL is optionally substituted heteroC949alkenyl. In some embodiments, RL is optionally substituted heteroC948alkenyl. In some embodiments, RL is optionally substituted heteroC9_17alkenyl. In some embodiments, RL is optionally substituted heteroC946alkenyl. In some embodiments, RL is optionally substituted heteroC945alkenyl. In some embodiments, RL is optionally substituted heteroC944alkenyl. In some embodiments, RL is optionally substituted heteroC943alkenyl. In some embodiments, RL is optionally substituted heteroC942alkenyl. In some embodiments, RL is optionally substituted heteroC9_1ialkenyl. In some embodiments, RL is optionally substituted heteroC940alkenyl.
[0265] In some embodiments, RL is optionally substituted heteroCio-soalkenyl. In some embodiments, RL is optionally substituted heteroCio-4oalkenyl. In some embodiments, RL is optionally substituted heteroCio-3oalkenyl. In some embodiments, RL is optionally substituted heteroCio-2oalkenyl. In some embodiments, RL is optionally substituted heteroCio-Nalkenyl. In some embodiments, RL is optionally substituted heteroC10-18alkenyl. In some embodiments, RL
is optionally substituted heteroCio_ralkenyl. In some embodiments, RL is optionally substituted heteroCio-malkenyl. In some embodiments, RL is optionally substituted heteroCio-isalkenyl. In ST 1 `sumuupoquio mos ui liCuo3upcipoiolou pouupsqns iCipuop 1 do ST `sumuupoquio = =
mos ui liCuo3Tiptipoiolou pouupsqns iCipuopdo ST _Ii `sumuupoquio mos ui liCuo3HEEIDoiolou pouupsqns iCipuopdo ST _Ii `sumuupoquio mos ui liCuo3upzipoiolou pouupsqns iCipuopdo ST, `sumuupoquio mos ui liCuo3HEITDoiolou pouupsqns iCipuopdo ST
`sumuupoquio = = 1 mos ui liCuo3TIE0IDoiolou pouupsqns iCipuopdo ST _Ii `sumuupoquio mos ui liCuo3HE6D010lou pouupsqns iCipuopdo ST _Ii `sumuupoquio mos ui liCuo3upspoiolou pouupsqns iCipuopdo ST
1 `sumuupoquio mos ui liCuo3HELDwolou pouupsqns iCipuopdo ST `sumuupoquio = = 1 mos ui liCuo3HE9Doiolou pouupsqns iCipuopdo ST _Ii `sumuupoquio mos ui 189Z0]
=pCuo3HEET-ZIDalopti pouupsqns iCipuopdo sT
`sumuupoquio mos ui liCuo3upti-zipoiolou poisupsqns iCipuopdo ST _Ii `sumuupoquio mos uI *IiCuo3HP"-zIpoiolou pouupsqns iCipuopdo ST _Ii `sumuupoquio mos ui liCuo3HE9I-zipoiolou pouupsqns iCipuopdo ST _Ii `sumuupoquio mos ui =pcuo3HELT-ziponloti pouupsqns iCipuopdo sT
_Ii `sumuupoquio mos ui liCuo3uppoiolou poisupsqns iCipuopdo ST _Ii `sumuupoquio mos uI *IiCuo3IIP6i-ziDwoloti pouupscins iCipuopdo ST I-N `sumuupoquio mos ui .pcuo3upoz-zip0iolou pouupsqns iCipuopdo ST I-N `sumuupoquio mos ui .pcuo3upoE-zipoiolou pouupsqns iCipuopdo ST II 1 `sumuupoquio mos ui .pcu331-pot-zipoiolou pouupsqns iCipuop 1 do ST II `sumuupoquio = =
mos ui liCuo3upoc-zipoiolou pouuluscins iCipuopdo ST I-N `sumuupoquio mos ui IL9Z0]
. I/W.03ff z I-I I D alopti pouupsqns iCipuopdo ST _Ii `sumuupoquio mos ui liCuoTBEI-IIDoiolou pouupsqns iCipuopdo sT
_Ii `sumuupoquio mos ui liCuo3upti-IIDoiolou poisupsqns iCipuopdo ST _Ii `sumuupoquio mos uI *IiCuo3IIP"-"Doiolou pouupsqns iCipuopdo ST _Ii `sumuupoquio mos ui 11Cuo3up9I-IIDalopti pouupsqns iCipuopdo ST I-N `sumuupoquio mos ui licUOTBL I-I I Dampti pouupsqns iCipuopdo sT
_Ii `sluouupoquio mos ui liCuo3upsi-IIDoiolou poisupsqns iCipuopdo ST _Ii `sumuupoquio mos uI 1JC11.33TIP61-TIDwoloti pouupscins iCipuopdo ST I-N `sumuupoquio mos ui .pcuo3upoz-iipoiolo1i pouupsqns iCipuopdo ST _Ii `sumuupoquio mos ui liCuo3upoE-IIDoiolo4 pouupsqns iCipuopdo ST II 1 `sumuupoquio mos ui .pcuo3up017-Iipoiolou pouupsqns iCipuop 1 do ST II `sumuupoquio = =
mos ui licUOTBOS-I iDoiolou pouuluscins iCipuopdo ST 1-N `sumuupoquio mos ui 199Z0]
.pcuo3upii-oiDwolo1i pouupscins iCipuopdo ST 1-N `sumuupoquio mos ui .pcuo3upzi-0iDoiolo1i pouupsqns iCipuopdo ST _Ii `sumuupoquio mos ui =pcuo3upI-0IDampti pouupsqns iCipuopdo sT
I-N `sumuupoquio mos ui lictIO3upti-0IDwolo1i poisupscins iCipuopdo ST
`sumuupoquio mos 6LI90/tIOZSI1IIDd L9tI90/SIOZ OM

optionally substituted heteroCi6alkenyl. In some embodiments, RL is optionally substituted heteroC ralkenyl. In some embodiments, RL is optionally substituted heteroCi8alkenyl. In some embodiments, RL is optionally substituted heteroCi9alkenyl. In some embodiments, RL is optionally substituted heteroC20alkenyl.
[0269] In some embodiments, for example, in any of the above embodiments, RL is a substituted heteroalkenyl group. In some embodiments, RL is an unsubstituted heteroalkenyl group. In some embodiments, RL is an optionally substituted straight-chain heteroalkenyl group.
In some embodiments, RL is a substituted straight-chain heteroalkenyl group.
In some embodiments, RL is an unsubstituted straight-chain heteroalkenyl group. In some embodiments, RL is an optionally substituted branched heteroalkenyl group. In some embodiments, RL is a substituted branched heteroalkenyl group. In some embodiments, RL is an unsubstituted branched heteroalkenyl group.
[0270] In some embodiments, RL is optionally substituted heteroC2_50alkynyl. In some embodiments, RL is optionally substituted heteroC2_40alkynyl. In some embodiments, RL is optionally substituted heteroC2_30alkynyl. In some embodiments, RL is optionally substituted heteroC2_20alkynyl. In some embodiments, RL is optionally substituted heteroC2_19alkynyl. In some embodiments, RL is optionally substituted heteroC2_18alkynyl. In some embodiments, RL is optionally substituted heteroC2_17alkynyl. In some embodiments, RL is optionally substituted heteroC2_16alkynyl. In some embodiments, RL is optionally substituted heteroC2_15alkynyl. In some embodiments, RL is optionally substituted heteroC2_14alkynyl. In some embodiments, RL is optionally substituted heteroC2_13alkynyl. In some embodiments, RL is optionally substituted heteroC2_12alkynyl. In some embodiments, RL is optionally substituted heteroC2_iialkynyl. In some embodiments, RL is optionally substituted heteroC2_10alkynyl. In some embodiments, RL is optionally substituted heteroC2_9alkynyl. In some embodiments, RL is optionally substituted heteroC2_8alkynyl. In some embodiments, RL is optionally substituted heteroC2_7alkynyl. In some embodiments, RL is optionally substituted heteroC2_6alkynyl.
[0271] In some embodiments, RL is optionally substituted heteroC4_50alkynyl. In some embodiments, RL is optionally substituted heteroC4_40alkynyl. In some embodiments, RL is optionally substituted heteroC4_30alkynyl. In some embodiments, RL is optionally substituted heteroC4_20alkynyl. In some embodiments, RL is optionally substituted heteroC4_19alkynyl. In some embodiments, RL is optionally substituted heteroC4_18alkynyl. In some embodiments, RL is optionally substituted heteroC4_17alkynyl. In some embodiments, RL is optionally substituted heteroC4_16alkynyl. In some embodiments, RL is optionally substituted heteroC4_15alkynyl. In some embodiments, RL is optionally substituted heteroC4_14alkynyl. In some embodiments, RL is optionally substituted heteroC4_13alkynyl. In some embodiments, RL is optionally substituted heteroC4_12alkynyl. In some embodiments, RL is optionally substituted heteroC4_iialkynyl. In some embodiments, RL is optionally substituted heteroC4_10alkynyl. In some embodiments, RL is optionally substituted heteroC4_9alkynyl. In some embodiments, RL is optionally substituted heteroC4_8alkynyl. In some embodiments, RL is optionally substituted heteroC4_7alkynyl. In some embodiments, RL is optionally substituted heteroC4_6alkynyl.
[0272] In some embodiments, RL is optionally substituted heteroC6_50alkynyl. In some embodiments, RL is optionally substituted heteroC6_40alkynyl. In some embodiments, RL is optionally substituted heteroC6_30alkynyl. In some embodiments, RL is optionally substituted heteroC6_20alkynyl. In some embodiments, RL is optionally substituted heteroC6_19alkynyl. In some embodiments, RL is optionally substituted heteroC6_18alkynyl. In some embodiments, RL is optionally substituted heteroC6_17alkynyl. In some embodiments, RL is optionally substituted heteroC6_16alkynyl. In some embodiments, RL is optionally substituted heteroC6_15alkynyl. In some embodiments, RL is optionally substituted heteroC6_14alkynyl. In some embodiments, RL is optionally substituted heteroC6_13alkynyl. In some embodiments, RL is optionally substituted heteroC6_12alkynyl. In some embodiments, RL is optionally substituted heteroC6_iialkynyl. In some embodiments, RL is optionally substituted heteroC6_10alkynyl. In some embodiments, RL is optionally substituted heteroC6_9alkynyl. In some embodiments, RL is optionally substituted heteroC6_8alkynyl. In some embodiments, RL is optionally substituted heteroC6_7alkynyl.
[0273] In some embodiments, RL is optionally substituted heteroC8_50alkynyl. In some embodiments, RL is optionally substituted heteroC8_40alkynyl. In some embodiments, RL is optionally substituted heteroC8_30alkynyl. In some embodiments, RL is optionally substituted heteroC8_20alkynyl. In some embodiments, RL is optionally substituted heteroC8_19alkynyl. In some embodiments, RL is optionally substituted heteroC8_18alkynyl. In some embodiments, RL is optionally substituted heteroC8_17alkynyl. In some embodiments, RL is optionally substituted heteroC8_16alkynyl. In some embodiments, RL is optionally substituted heteroC8_15alkynyl. In some embodiments, RL is optionally substituted heteroC8_14alkynyl. In some embodiments, RL is optionally substituted heteroC8_13alkynyl. In some embodiments, RL is optionally substituted heteroC8_12alkynyl. In some embodiments, RL is optionally substituted heteroC8_iialkynyl. In some embodiments, RL is optionally substituted heteroC8_10alkynyl. In some embodiments, RL is optionally substituted heteroC8_9alkynyl.
[0274] In some embodiments, RL is optionally substituted heteroC9_50alkynyl. In some embodiments, RL is optionally substituted heteroC9_40alkynyl. In some embodiments, RL is optionally substituted heteroC9_30alkynyl. In some embodiments, RL is optionally substituted heteroC9_20alkynyl. In some embodiments, RL is optionally substituted heteroC9_19alkynyl. In some embodiments, RL is optionally substituted heteroC9_18alkynyl. In some embodiments, RL is optionally substituted heteroC9_17alkynyl. In some embodiments, RL is optionally substituted heteroC9_16alkynyl. In some embodiments, RL is optionally substituted heteroC9_15alkynyl. In some embodiments, RL is optionally substituted heteroC9_14alkynyl. In some embodiments, RL is optionally substituted heteroC9_13alkynyl. In some embodiments, RL is optionally substituted heteroC9_12alkynyl. In some embodiments, RL is optionally substituted heteroC9_iialkynyl. In some embodiments, RL is optionally substituted heteroC9_10alkynyl.
[0275] In some embodiments, RL is optionally substituted heteroCio-5oalkynyl. In some embodiments, RL is optionally substituted heteroCio-4oalkynyl. In some embodiments, RL is optionally substituted heteroCio-3oalkynyl. In some embodiments, RL is optionally substituted heteroCio-2oalkynyl. In some embodiments, RL is optionally substituted heteroCio-Nalkynyl. In some embodiments, RL is optionally substituted heteroCio-isalkynyl. In some embodiments, RL
is optionally substituted heteroC io-ralkynyl. In some embodiments, RL is optionally substituted heteroCio-malkynyl. In some embodiments, RL is optionally substituted heteroC10-15alkynyl. In some embodiments, RL is optionally substituted heteroCio-i4alkynyl. In some embodiments, RL
is optionally substituted heteroCio-Dalkynyl. In some embodiments, RL is optionally substituted heteroCio-ualkynyl. In some embodiments, RL is optionally substituted heteroCio-iialkynyl.
[0276] In some embodiments, RL is optionally substituted heteroC1i-5oalkynyl. In some embodiments, RL is optionally substituted heteroC1i-4oalkynyl. In some embodiments, RL is optionally substituted heteroC1i-3oalkynyl. In some embodiments, RL is optionally substituted heteroC1i-2oalkynyl. In some embodiments, RL is optionally substituted heteroCii-Nalkynyl. In some embodiments, RL is optionally substituted heteroCii-isalkynyl. In some embodiments, RL
is optionally substituted heteroCii_ralkynyl. In some embodiments, RL is optionally substituted heteroCii-malkynyl. In some embodiments, RL is optionally substituted heteroCii-isalkynyl. In some embodiments, RL is optionally substituted heteroCii-i4alkynyl. In some embodiments, RL
is optionally substituted heteroCii-Dalkynyl. In some embodiments, RL is optionally substituted hetero C ii-ualkynyl.
[0277] In some embodiments, RL is optionally substituted heteroCi2-5oalkynyl. In some embodiments, RL is optionally substituted heteroCi2-4oalkynyl. In some embodiments, RL is optionally substituted heteroCi2-3oalkynyl. In some embodiments, RL is optionally substituted heteroCi2-2oalkynyl. In some embodiments, RL is optionally substituted heteroCi2-19alkynyl. In some embodiments, RL is optionally substituted heteroCi2-18alkynyl. In some embodiments, RL
is optionally substituted heteroCi2-ralkynyl. In some embodiments, RL is optionally substituted heter0C12-16alkynyl. In some embodiments, RL is optionally substituted heteroC12-15alkynyl. In some embodiments, RL is optionally substituted heteroCi2-14alkynyl. In some embodiments, RL
is optionally substituted heteroC12-13alkynyl.
[0278] In some embodiments, RL is optionally substituted heteroC6alkynyl.
In some embodiments, RL is optionally substituted heteroC7alkynyl. In some embodiments, RL is optionally substituted heteroC8alkynyl. In some embodiments, RL is optionally substituted heteroC9alkynyl. In some embodiments, RL is optionally substituted heteroCi0alkynyl. In some embodiments, RL is optionally substituted heteroCiialkynyl. In some embodiments, RL is optionally substituted heteroCi2alkynyl. In some embodiments, RL is optionally substituted heteroC nalkynyl. In some embodiments, RL is optionally substituted heteroCi4alkynyl. In some embodiments, RL is optionally substituted heteroCi5alkynyl. In some embodiments, RL is optionally substituted heteroCi6alkynyl. In some embodiments, RL is optionally substituted heteroC ralkynyl. In some embodiments, RL is optionally substituted heteroCi8alkynyl. In some embodiments, RL is optionally substituted heteroCi9alkynyl. In some embodiments, RL is optionally substituted heteroC20alkynyl.
[0279] In some embodiments, for example, in any of the above embodiments, RL is a substituted heteroalkynyl group. In some embodiments, RL is an unsubstituted heteroalkynyl group. In some embodiments, RL is an optionally substituted straight-chain heteroalkynyl group.

In some embodiments, RL is a substituted straight-chain heteroalkynyl group.
In some embodiments, RL is an unsubstituted straight-chain heteroalkynyl group. In some embodiments, RL is an optionally substituted branched heteroalkynyl group. In some embodiments, RL is a substituted branched heteroalkynyl group. In some embodiments, RL is an unsubstituted branched heteroalkynyl group.
[0280] In some embodiments, RL is a polymer. As used herein, a "polymer", in some embodiments, refers to a compound comprised of at least 3 (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.) repeating covalently bound structural units. The polymer is in certain embodiments biocompatible (i.e., non-toxic). Exemplary polymers include, but are not limited to, cellulose polymers (e.g., hydroxyethylcellulose, ethylcellulose, carboxymethylcellulose, methylc cellulose, hydroxypropylmethylcellulose (HPMC)), dextran polymers, polymaleic acid polymers, poly(acrylic acid) polymers, poly(vinylalcohol) polymers, polyvinylpyrrolidone (PVP) polymers, and polyethyleneglycol (PEG) polymers, and combinations thereof [0281] In some embodiments, RL is a lipophilic, hydrophobic and/or non-polar group. In some embodiments, RL is a lipophilic group. In some embodiments, RL is a hydrophobic group.
In some embodiments, RL is a non-polar group.
[0282] In some embodiments, when an RL group is depicted as bisecting a carbon-carbon bond, e.g., of the formula (i), it is understood that RL may be bonded to either carbon.
[0283] In some embodiments, at least one instance of RQ, R2, R6, or R7 is a group of the formula (i), (ii), or (iii). In some embodiments, at least one instance of R6 or R7 of Rl is a group of formula (i), (ii) or (iii). In some embodiments, at least one instance of R6 or R7 of Rl is a group of formula (i). In some embodiments, at least one instance of R6 or R7 of Rl is a group of formula (i-a). In some embodiments, at least one instance of R6 or R7 of Rl is a group of formula (i-al). In some embodiments, at least one instance of R6 or R7 of Rl is a group of formula (i-b).
In some embodiments, at least one instance of R6 or R7 of Rl is a group of formula (ii). In some embodiments, at least one instance of R6 or R7 of Rl is a group of formula (iii).
[0284] Various combinations of the above embodiments of Formula I are contemplated herein.

[0285] In some embodiments, wherein each instance of Q is 0, the compound of formula I is a compound of formula I-a:
R2 ip I-a or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.
[0286] In certain embodiments, at least one Rl is a group of formula (iv). In certain embodiments, each instance of Rl is a group of formula (iv). In certain embodiments, each instance of R2 is independently hydrogen or optionally substituted Ci_6alkyl.
In certain embodiments, each instance of R2 is hydrogen. In certain embodiments, at least one instance of R2 is a group of formula (i). In certain embodiments, at least one instance of R2 is a group of formula (ii). In certain embodiments, at least one instance of R2 is a group of formula (iii). In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3.
[0287] In some embodiments, wherein at least one Rl is a group of formula (iv), a compound of formula I is a compound of formula I-b:
R2 .1 I-b or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.
[0288] In certain embodiments, each instance of Rl is a group of formula (iv). In certain embodiments, each instance of R2 is independently hydrogen or optionally substituted Ci_6alkyl.
In certain embodiments, each instance of R2 is hydrogen. In certain embodiments, at least one instance of R2 is a group of formula (i). In certain embodiments, at least one instance of R2 is a group of formula (ii). In certain embodiments, at least one instance of R2 is a group of formula (iii). In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3. In certain embodiments, L is an optionally substituted alkylene. In certain embodiments, R6 is a group of formula (i). In certain embodiments, R6 is a group of formula (ii). In certain embodiments, R6 is a group of formula (iii). In certain embodiments, R7 is a group of formula (i). In certain embodiments, R7 is a group of formula (ii). In certain embodiments, R7 is a group of formula (iii). In certain embodiments, both R6 and R7 are independently groups of formula (i), (ii), or (iii).
[0289] In some embodiments, wherein each instance of Rl is a group the formula (iv), a compound of Formula I is a compound of formula I-c:

.R7 N¨L R2 or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.

[0290] In certain embodiments, each instance of R2 is independently hydrogen or optionally substituted Ci_6alkyl. In certain embodiments, each instance of R2 is hydrogen. In certain embodiments, at least one instance of R2 is a group of formula (i). In certain embodiments, at least one instance of R2 is a group of formula (ii). In certain embodiments, at least one instance of R2 is a group of formula (iii). In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3. In certain embodiments, L
is an optionally substituted alkylene. In certain embodiments, R6 is a group of formula (i). In certain embodiments, R6 is a group of formula (ii). In certain embodiments, R6 is a group of formula (iii). In certain embodiments, R7 is a group of formula (i). In certain embodiments, R7 is a group of formula (ii). In certain embodiments, R7 is a group of formula (iii). In certain embodiments, both R6 and R7 are independently groups of formula (i), (ii), or (iii).
[0291] In some embodiments, p=1. In some embodiments, a compound of formula I-c is a compound of formula I-cl:
R6 ,,R7 o I-ct or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.
[0292] In certain embodiments, each instance of R2 is independently hydrogen or optionally substituted Ci_6alkyl. In certain embodiments, each instance of R2 is hydrogen. In certain embodiments, at least one instance of R2 is a group of formula (i). In certain embodiments, at least one instance of R2 is a group of formula (ii). In certain embodiments, at least one instance of R2 is a group of formula (iii). In certain embodiments, L is an optionally substituted alkylene. In certain embodiments, R6 is a group of formula (i). In certain embodiments, R6 is a group of formula (ii). In certain embodiments, R6 is a group of formula (iii). In certain embodiments, R7 is a group of formula (i). In certain embodiments, R7 is a group of formula (ii). In certain embodiments, R7 is a group of formula (iii). In certain embodiments, both R6 and R7 are independently groups of formula (i), (ii), or (iii). In some embodiments, R6 and R7 are the same group of formula (i). In some embodiments, R6 and R7 are the same group of formula (i-a). In some embodiments, R6 and R7 are the same group of formula (i-al). In some embodiments, R6 and R7 are the same group of formula (i-b). In some embodiments, R6 and R7 are the same group of formula (ii). In some embodiments, R6 and R7 are the same group of formula (iii).
[0293] In some embodiments, each instance of R2 is hydrogen. In some embodiments, a compound of formula I-c is a compound of formula I-c2:

0 1-1\is;_\1----10 N¨L

I-c2 or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.
[0294] In certain embodiments, L is an optionally substituted alkylene.
In certain embodiments, R6 is a group of formula (i). In certain embodiments, R6 is a group of formula (ii).
In certain embodiments, R6 is a group of formula (iii). In certain embodiments, R7 is a group of formula (i). In certain embodiments, R7 is a group of formula (ii). In certain embodiments, R7 is a group of formula (iii). In certain embodiments, both R6 and R7 are independently groups of formula (i), (ii), or (iii). In some embodiments, R6 and R7 are the same group of formula (i). In some embodiments, R6 and R7 are the same group of formula (i-a). In some embodiments, R6 and R7 are the same group of formula (i-al). In some embodiments, R6 and R7 are the same group of formula (i-b). In some embodiments, R6 and R7 are the same group of formula (ii). In some embodiments, R6 and R7 are the same group of formula (iii).
[0295] In some embodiments, L is an optionally substituted alkylene. In some embodiments, a compound of formula I-c is a compound of formula I-c3:
çhq H Re Re NH
N ___________________________________ q I-c3 or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.
[0296] In some embodiments, q is an integer between 1 and 10, inclusive.
In certain embodiments, R6 is a group of formula (i). In certain embodiments, R6 is a group of formula (ii).
In certain embodiments, R6 is a group of formula (iii). In certain embodiments, R7 is a group of formula (i). In certain embodiments, R7 is a group of formula (ii). In certain embodiments, R7 is a group of formula (iii). In certain embodiments, both R6 and R7 are independently groups of formula (i), (ii), or (iii). In some embodiments, R6 and R7 are the same group of formula (i). In some embodiments, R6 and R7 are the same group of formula (i-a). In some embodiments, R6 and R7 are the same group of formula (i-al). In some embodiments, R6 and R7 are the same group of formula (i-b). In some embodiments, R6 and R7 are the same group of formula (ii). In some embodiments, R6 and R7 are the same group of formula (iii).

[0297] In some embodiments, a compound of formula I is a compound of formula I-d:
HO
RL
______________________________________________ 'RL
"-OH
N=c 0)_ ____________________________________ 0 FZ2 p RL N
HO-OH
I-d wherein each of p, R2 and RI- is independently as defined above and described herein.
[0298] In some embodiments, a compound of formula I is a compound of formula I-e:
HO
RL
RL
N-µz _______________________________________________ '-OH
R2 _____________________________________ RL N
HO-<RL
OH
I-e wherein each of R2 and RI- is independently as defined above and described herein.

[0299] In some embodiments, a compound of formula I is a compound of formula I-f:
HO

¨R1-R2 / 7¨)_ RL OH
\14 ____________________________________ 1-0 1\1, RV¨N
HO
-.-R1-OH , I-f wherein each of R2 and RI- is independently as defined above and described herein.
[0300] In some embodiments, provided liposomes include a cationic lipid described in WO 2013063468 and in U.S. provisional application entitled "Lipid Formulations for Delivery of Messenger RNA" filed concurrently with the present application on even date, both of which are incorporated by reference herein. In some embodiments, a compound of formula I
is a compound of formula I-c I -a:
RL C)\ RI-RI Ry H H r j1N' R' R' R2 ) \ q N
_ 0¨ 0 N
\
( )(i R2 R' R' H0)7( NOH
R' R' RL RL I-cl -a or a pharmaceutically acceptable salt thereof, wherein:
each R2 independently is hydrogen or C1_3 alkyl;
each q independently is 2 to 6;
each R' independently is hydrogen or C1_3 alkyl;
and each RL independently is C8_12 alkyl.
[0301] In some embodiments, each R2 independently is hydrogen, methyl or ethyl. In some embodiments, each R2 independently is hydrogen or methyl. In some embodiments, each R2 is hydrogen.
[0302] In some embodiments, each q independently is 3 to 6. In some embodiments, each q independently is 3 to 5. In some embodiments, each q is 4.
[0303] In some embodiments, each R' independently is hydrogen, methyl or ethyl. In some embodiments, each R' independently is hydrogen or methyl. In some embodiments, each R' independently is hydrogen.
[0304] In some embodiments, each RL independently is C8_12 alkyl. In some embodiments, each RL independently is n-C8_12 alkyl. In some embodiments, each RL
independently is C9_11 alkyl. In some embodiments, each RL independently is n-C9_11 alkyl. In some embodiments, each RL independently is Cio alkyl. In some embodiments, each RL
independently is n-C10 alkyl.
[0305] In some embodiments, each R2 independently is hydrogen or methyl;
each q independently is 3 to 5; each R' independently is hydrogen or methyl; and each RL independently is C8_12 alkyl.
[0306] In some embodiments, each R2 is hydrogen; each q independently is 3 to 5; each R' is hydrogen; and each RL independently is C8_12 alkyl.
[0307] In some embodiments, each R2 is hydrogen; each q is 4; each R' is hydrogen; and each RL independently is C8_12 alkyl.
[0308] In some embodiments, a compound of formula I is a compound of formula I-g:

HO
/N-_OH
RL
RL _____________________________ HN
CI-NH
____________________________ N) HO
OH
I-g wherein each of RI- is independently as defined above and described herein.
[0309] In some embodiments, a compound of formula I is a compound of formula X:
HO
C uH2 -zaz( /-HOD\ ) C 1'6 1 X
or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.
[0310] In some embodiments, a compound of formula I is a compound of formula X-1:

HO
CT.
N¨vC1oH21 i\¨OH
R2 _____________________________________ \N
01_ 0 C101-121N,¨N
HO¨/
CioH21 OH

or a pharmaceutically acceptable salt thereof, wherein each R2 is independently as defined above and described herein.
[0311] In some embodiments, a compound of formula I is HO
/ OH
/ n r HN
i¨NH
n-C10H21/ N
HO _________________________ V
OH
or a pharmaceutically acceptable salt thereof [0312] Additional examples of cationic lipids suitable for the present invention are described in WO 2013063468, which is incorporated by reference herein in its entirety.

Chemical definitions [0313] Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001;
Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.
[0314] Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers.
For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977);
Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S.H. Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The invention additionally contemplates compounds as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.
[0315] When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example "C1-6 alkyl" is intended to encompass, Cl, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6 alkyl.
[0316] As used herein, "alkyl" refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 50 carbon atoms ("C1-50 alkyl"). In some embodiments, an alkyl group has 1 to 40 carbon atoms ("C1-40 alkyl"). In some embodiments, an alkyl group has 1 to 30 carbon atoms ("C1-30 alkyl"). In some embodiments, an alkyl group has 1 to 20 carbon atoms ("C1-20 alkyl"). In some embodiments, an alkyl group has 1 to 10 carbon atoms ("C1-10 alkyl"). In some embodiments, an alkyl group has 1 to 9 carbon atoms ("C1-9 alkyl"). In some embodiments, an alkyl group has 1 to 8 carbon atoms ("C1-8 alkyl"). In some embodiments, an alkyl group has 1 to 7 carbon atoms ("C1-7 alkyl"). In some embodiments, an alkyl group has 1 to 6 carbon atoms ("C1-6 alkyl"). In some embodiments, an alkyl group has 1 to 5 carbon atoms ("C1-5 alkyl"). In some embodiments, an alkyl group has 1 to 4 carbon atoms ("C1-4 alkyl"). In some embodiments, an alkyl group has 1 to 3 carbon atoms ("C1-3 alkyl"). In some embodiments, an alkyl group has 1 to 2 carbon atoms ("C1-2 alkyl"). In some embodiments, an alkyl group has 1 carbon atom ("Cl alkyl").
In some embodiments, an alkyl group has 2 to 6 carbon atoms ("C2-6 alkyl"). Examples of C1-6 alkyl groups include, without limitation, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methy1-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8) and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an "unsubstituted alkyl") or substituted (a "substituted alkyl") with one or more substituents. In certain embodiments, the alkyl group is an unsubstituted C1-50 alkyl. In certain embodiments, the alkyl group is a substituted C1-50 alkyl.
[0317] As used herein, "heteroalkyl" refers to an alkyl group as defined herein which further includes at least one heteroatom (e.g., 1 to 25, e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 50 carbon atoms and 1 or more heteroatoms within the parent chain ("heteroC1-50 alkyl"). In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 40 carbon atoms and 1 or more heteroatoms within the parent chain ("heteroC1-40 alkyl"). In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 30 carbon atoms and 1 or more heteroatoms within the parent chain ("heteroC1-30 alkyl"). In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 20 carbon atoms and 1 or more heteroatoms within the parent chain ("heteroC1-20 alkyl"). In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 10 carbon atoms and 1 or more heteroatoms within the parent chain ("heteroC1-10 alkyl"). In some embodiments, a heteroalkyl group is a saturated group having 1 to 9 carbon atoms and 1 or more heteroatoms within the parent chain ("heteroC1-9 alkyl"). In some embodiments, a heteroalkyl group is a saturated group having 1 to 8 carbon atoms and 1 or more heteroatoms within the parent chain ("heteroC1-8 alkyl"). In some embodiments, a heteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1 or more heteroatoms within the parent chain ("heteroC1-7 alkyl"). In some embodiments, a heteroalkyl group is a saturated group having 1 to 6 carbon atoms and 1 or more heteroatoms within the parent chain ("heteroC1-6 alkyl"). In some embodiments, a heteroalkyl group is a saturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms within the parent chain ("heteroC1-5 alkyl"). In some embodiments, a heteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1 or 2 heteroatoms within the parent chain ("heteroC1-4 alkyl"). In some embodiments, a heteroalkyl group is a saturated group having 1 to 3 carbon atoms and 1 heteroatom within the parent chain ("heteroC1-3 alkyl"). In some embodiments, a heteroalkyl group is a saturated group having 1 to 2 carbon atoms and 1 heteroatom within the parent chain ("heteroC1-2 alkyl"). In some embodiments, a heteroalkyl group is a saturated group having 1 carbon atom and 1 heteroatom ("heteroC1 alkyl").
In some embodiments, a heteroalkyl group is a saturated group having 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parent chain ("heteroC2-6 alkyl"). Unless otherwise specified, each instance of a heteroalkyl group is independently unsubstituted (an "unsubstituted heteroalkyl") or substituted (a "substituted heteroalkyl") with one or more substituents. In certain embodiments, the heteroalkyl group is an unsubstituted heteroC1-50 alkyl. In certain embodiments, the heteroalkyl group is a substituted heteroC1-50 alkyl.
[0318] As used herein, "alkenyl" refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 50 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds) ("C2-50 alkenyl"). In some embodiments, an alkenyl group has 2 to 40 carbon atoms ("C2-40 alkenyl"). In some embodiments, an alkenyl group has 2 to 30 carbon atoms ("C2-30 alkenyl"). In some embodiments, an alkenyl group has 2 to 20 carbon atoms ("C2-20 alkenyl"). In some embodiments, an alkenyl group has 2 to 10 carbon atoms ("C2-10 alkenyl"). In some embodiments, an alkenyl group has 2 to 9 carbon atoms ("C2-9 alkenyl").
In some embodiments, an alkenyl group has 2 to 8 carbon atoms ("C2-8 alkenyl"). In some embodiments, an alkenyl group has 2 to 7 carbon atoms ("C2-7 alkenyl"). In some embodiments, an alkenyl group has 2 to 6 carbon atoms ("C2-6 alkenyl"). In some embodiments, an alkenyl group has 2 to 5 carbon atoms ("C2-5 alkenyl"). In some embodiments, an alkenyl group has 2 to 4 carbon atoms ("C2-4 alkenyl").
In some embodiments, an alkenyl group has 2 to 3 carbon atoms ("C2-3 alkenyl"). In some embodiments, an alkenyl group has 2 carbon atoms ("C2 alkenyl"). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-buteny1). Examples of C2-4 alkenyl groups include, without limitation, ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-6 alkenyl groups include the aforementioned C2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like.
Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted (an "unsubstituted alkenyl") or substituted (a "substituted alkenyl") with one or more substituents. In certain embodiments, the alkenyl group is an unsubstituted C2-50 alkenyl. In certain embodiments, the alkenyl group is a substituted C2-50 alkenyl.
[0319] As used herein, "heteroalkenyl" refers to an alkenyl group as defined herein which further includes at least one heteroatom (e.g., 1 to 25, e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkenyl group refers to a group having from 2 to 50 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain ("heteroC2-50 alkenyl"). In certain embodiments, a heteroalkenyl group refers to a group having from 2 to 40 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain ("heteroC2-40 alkenyl"). In certain embodiments, a heteroalkenyl group refers to a group having from 2 to 30 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain ("heteroC2-30 alkenyl"). In certain embodiments, a heteroalkenyl group refers to a group having from 2 to 20 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain ("heteroC2-20 alkenyl"). In certain embodiments, a heteroalkenyl group refers to a group having from 2 to 10 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain ("heteroC2-10 alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 9 carbon atoms at least one double bond, and 1 or more heteroatoms within the parent chain ("heteroC2-9 alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 8 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain ("heteroC2-8 alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 7 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain ("heteroC2-7 alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain ("heteroC2-6 alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain ("heteroC2-5 alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 4 carbon atoms. at least one double bond, and for 2 heteroatoms within the parent chain ("heteroC2-4 alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain ("heteroC2-3 alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double, bond, and 1 or 2 heteroatoms within the parent chain ("heteroC2-6 alkenyl"). Unless otherwise specified, each instance of a heteroalkenyl group is independently unsubstituted (an "unsubstituted heteroalkenyl") or substituted (a "substituted heteroalkenyl") with one or more substituents. In certain embodiments, the heteroalkenyl group is an unsubstituted heteroC2-50 alkenyl. In certain embodiments, the heteroalkenyl group is a substituted heteroC2-50 alkenyl.
[0320] As used herein, "alkynyl" refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 50 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) and optionally one or more double bonds (e.g., 1, 2, 3, or 4 double bonds) ("C2-50 alkynyl"). An alkynyl group that has one or more triple bonds and one or more double bonds is also referred to as an "ene-yne". In some embodiments, an alkynyl group has 2 to 40 carbon atoms ("C2-40 alkynyl"). In some embodiments, an alkynyl group has 2 to 30 carbon atoms ("C2-30 alkynyl"). In some embodiments, an alkynyl group has 2 to 20 carbon atoms ("C2-20 alkynyl"). In some embodiments, an alkynyl group has 2 to 10 carbon atoms ("C2-10 alkynyl").
In some embodiments, an alkynyl group has 2 to 9 carbon atoms ("C2-9 alkynyl"). In some embodiments, an alkynyl group has 2 to 8 carbon atoms ("C2-8 alkynyl"). In some embodiments, an alkynyl group has 2 to 7 carbon atoms ("C2-7 alkynyl"). In some embodiments, an alkynyl group has 2 to 6 carbon atoms ("C2-6 alkynyl"). In some embodiments, an alkynyl group has 2 to 5 carbon atoms ("C2-5 alkynyl"). In some embodiments, an alkynyl group has 2 to 4 carbon atoms ("C2-4 alkynyl").
In some embodiments, an alkynyl group has 2 to 3 carbon atoms ("C2-3 alkynyl"). In some embodiments, an alkynyl group has 2 carbon atoms ("C2 alkynyl"). The one or more carbon--carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butyny1). Examples of C2-4 alkynyl groups include, without limitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C2-6 alkenyl groups include the aforementioned C2-4 alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (an "unsubstituted alkynyl") or substituted (a "substituted alkynyl") with one or more substituents. In certain embodiments, the alkynyl group is an unsubstituted C2-50 alkynyl.
In certain embodiments, the alkynyl group is a substituted C2-50 alkynyl.
[0321] As used herein, "heteroalkynyl" refers to an alkynyl group as defined herein which further includes at least one heteroatom (e.g., 1 to 25, e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkynyl group refers to a group having from 2 to 50 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain ("heteroC2-50 alkynyl"). In certain embodiments, a heteroalkynyl group refers to a group having from 2 to 40 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain ("heteroC2-40 alkynyl"). In certain embodiments, a heteroalkynyl group refers to a group having from 2 to 30 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain ("heteroC2-30 alkynyl"). In certain embodiments, a heteroalkynyl group refers to a group having from 2 to 20 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain ("heteroC2-20 alkynyl"). In certain embodiments, a heteroalkynyl group refers to a group having from 2 to 10 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain ("heteroC2-10 alkynyl"). In some embodiments, a heteroalkynyl group has 2 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain ("heteroC2-9 alkynyl"). In some embodiments, a heteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain ("heteroC2-8 alkynyl"). In some embodiments, a heteroalkynyl group has 2 to 7 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain ("heteroC2-7 alkynyl"). In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain ("heteroC2-6 alkynyl"). In some embodiments, a heteroalkynyl group has 2 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain ("heteroC2-5 alkynyl"). In some embodiments, a heteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond, and for 2 heteroatoms within the parent chain ("heteroC2-4 alkynyl"). In some embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain ("heteroC2-3 alkynyl"). In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond. and 1 or 2 heteroatoms within the parent chain ("heteroC2-6 alkynyl"). Unless otherwise specified, each instance of a heteroalkynyl group is independently unsubstituted (an "unsubstituted heteroalkynyl") or substituted (a "substituted heteroalkynyl") with one or more substituents. In certain embodiments, the heteroalkynyl group is an unsubstituted heteroC2-50 alkynyl. In certain embodiments, the heteroalkynyl group is a substituted heteroC2-50 alkynyl.
[0322] As used herein, "carbocyclyl" or "carbocyclic" refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms ("C3-10 carbocyclyl") and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms ("C3-8 carbocyclyl"). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms ("C3-7 carbocyclyl"). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms ("C3-6 carbocyclyl"). In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms ("C4-6 carbocyclyl"). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms ("C5-6 carbocyclyl"). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms ("C5-10 carbocyclyl"). Exemplary C3-6 carbocyclyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like.
Exemplary C3-8 carbocyclyl groups include, without limitation, the aforementioned C3-6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (C8), and the like.
Exemplary C3-10 carbocyclyl groups include, without limitation, the aforementioned C3-8 carbocyclyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decanyl (C10), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic ("monocyclic carbocyclyl") or polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system ("bicyclic carbocyclyl") or tricyclic system ("tricyclic carbocyclyl")) and can be saturated or can contain one or more carbon-carbon double or triple bonds. "Carbocycly1"
also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted (an "unsubstituted carbocyclyl") or substituted (a "substituted carbocyclyl") with one or more substituents. In certain embodiments, the carbocyclyl group is an unsubstituted C3-10 carbocyclyl. In certain embodiments, the carbocyclyl group is a substituted C3-10 carbocyclyl.
[0323] In some embodiments, "carbocyclyl" or "carbocyclic" is referred to as a "cycloalkyl", i.e., a monocyclic, saturated carbocyclyl group having from 3 to 10 ring carbon atoms ("C3-10 cycloalkyl"). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms ("C3-8 cycloalkyl"). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms ("C3-6, cycloalkyl"). In some embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms ("C4-6 cycloalkyl"). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms ("C5-6 cycloalkyl"). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms ("C5-10 cycloalkyl"). Examples of C5-6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5).
Examples of C3-6 cycloalkyl groups include the aforementioned C5-6 cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-8 cycloalkyl groups include the aforementioned C3-6 cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an "unsubstituted cycloalkyl") or substituted (a "substituted cycloalkyl") with one or more substituents. In certain embodiments, the cycloalkyl group is an unsubstituted C3-10 cycloalkyl. In certain embodiments, the cycloalkyl group is a substituted C3-10 cycloalkyl.

[0324] As used herein, "heterocyclyl" or "heterocyclic" refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("3-14 membered heterocyclyl"). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A
heterocyclyl group can either be monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system ("bicyclic heterocyclyl") or tricyclic system ("tricyclic heterocyclyl")). and can be saturated or can contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings.
"Heterocycly1" also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of heterocyclyl is independently unsubstituted (an "unsubstituted heterocyclyl") or substituted (a "substituted heterocyclyl") with one or more substituents. In certain embodiments, the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl.
[0325] In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-10 membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-8 membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-6 membered heterocyclyl"). In some embodiments, the 5-6 membered heterocyclyl has 1 or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heterocyclyl has 1 or 2 ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.

[0326] Exemplary 3-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl.
Exemplary 5-membered heterocyclyl groups containing 1 heteroatom include, without limitation.
tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrroly1-2,5-dione. Exemplary 5- membered heterocyclyl groups containing 2 heteroatoms include, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, pip erazinyl, morpholinyl, dithianyl, dioxanyl.
Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl.
Exemplary bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b] pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b ]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo-[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno [3,2- b]pyridinyl, 1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.
[0327] As used herein, "aryl" refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 7C electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system ("C6-14 aryl"). In some embodiments, an aryl group has 6 ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C10 aryl"; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("C14 aryl"; e.g., anthracyl).
"Aryl" also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an "unsubstituted aryl") or substituted (a "substituted aryl") with one or more substituents. In certain embodiments, the aryl group is an unsubstituted C6-14 aryl. In certain embodiments, the aryl group is a substituted C6-14 aryl.
[0328] As used herein, "heteroaryl" refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 7C electrons shared in a cyclic array) having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4 ring heteroatoms) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-14 membered heteroaryl"). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system.
"Heteroaryl" also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system.
Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indoly1) or the ring that does not contain a heteroatom (e.g., 5-indoly1).
[0329] In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-10 membered heteroaryl"). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-8 membered heteroaryl"). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-6 membered heteroaryl"). In some embodiments, the 5-6 membered heteroaryl has 1 or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heteroaryl has 1 or 2 ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted (an "unsubstituted heteroaryl") or substituted (a "substituted heteroaryl") with one or more substituents. In certain embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl.
[0330] Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, without limitation. pyridinyl.
Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl.
Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing 1 heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, b enzimidazo lyl, b enz oxazo lyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl and phenazinyl.
[0331] As used herein, the term "partially unsaturated" refers to a ring moiety that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic groups (e.g., aryl or heteroaryl moieties) as herein defined.
[0332] As used herein, the term "saturated" refers to a ring moiety that does not contain a double or triple bond, i.e., the ring contains all single bonds.

[0333] Affixing the suffix "-ene" to a group indicates the group is a divalent moiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene is the divalent moiety of alkenyl, [0334] alkynylene is the divalent moiety of alkynyl, heteroalkylene is the divalent moiety of heteroalkyl, heteroalkenylene is the divalent moiety of heteroalkenyl, heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclylene is the divalent moiety of carbocyclyl, heterocyclylene is the divalent moiety of heterocyclyl, arylene is the divalent moiety of aryl, and heteroarylene is the divalent moiety of heteroaryl.
[0335] As understood from the above, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are, in certain embodiments, optionally substituted. Optionally substituted refers to a group which may be substituted or unsubstituted (e.g., "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted"
heteroalkyl, "substituted" or "unsubstituted" heteroalkenyl, "substituted" or .'unsubstituted"
heteroalkynyl. "substituted" or "unsubstituted" carbocyclyl. "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl or "substituted" or "unsubstituted" heteroaryl group). In general, the term "substituted" means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position.
The term "substituted" is contemplated to include substitution with all permissible substituents of organic compounds, any of the substituents described herein that results in the formation of a stable compound. The present invention contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.
[0336] Exemplary carbon atom substituents include, but are not limited to, halogen, -CN, -NO2, -N3, -S02H, -S03H, -OH, -0Raa, -0N(Rbb)2, -N(Rbb)2, -N(Rbb)3+X-, -N(ORcc)Rbb, -SeH, -SeRaa, -SH, -SRaa, -SSRcc, -C(=0)Raa, -CO2H, -CHO, -C(ORcc)2, - CO2Raa, -OC(=0)Raa, -0CO2Raa, -C(=0)N(Rbb)2, -0C(=0)N(Rbb)2, -NRbbC(=0)Raa, -NRbbCO2Raa, -NRbbC(=0)N(Rbb)2, -C(=NRbb)Raa, -C(=NRbb)0Raa, -0C(=NRbb)Raa, - OC(=NRbb)0Raa, -C(=NRbb)N(Rbb)2, -0C(=NRbb)N(Rbb)2, -NRbbC(=NRbb)N(Rbb)2, - C(=0)NRbbSO2Raa, -NRbbSO2Raa, -SO2N(Rbb)2, -SO2Raa, -S020Raa, -0S02Raa, -S(=0)Raa, -0S(=0)Raa, -Si(Raa)3 -0Si(Raa)3 -C(=S)N(Rbb)2, -C(=0)SRaa, -C(=S)SRaa, - SC(=S)SRaa, -SC(=0)SRaa, -OC(=0)SRaa, -SC(=0)0Raa, -SC(=0)Raa, -P(=0)2Raa, -0P(=0)2Raa, -P(=0)(Raa)2, -OP(=0)(Raa)2, -0P(=0)(ORcc)2, -P(0)2N(Rbb)2, -OP(0)2N(Rbb)2, - P(=0)(NRbb)2, -OP(=0)(NRbb)2, -NRbbP(=0)(ORcc)2, -NRbbP(=0)(NRbb)2, -P(Rcc)2, - P(Rcc)3, -0P(Rcc)2, -OP(Rcc)3, -B(Raa)2, -B(ORcc)2, -BRaa(ORcc), C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-14 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;
[0337] or two geminal hydrogens on a carbon atom are replaced with the group =0, =S, =NN(Rbb)2, =NNRbbC(=0)Raa, =NNRbbC(=0)0Raa, =NNRbbS(=0)2Raa, =NRbb, or =NORcc;
[0338] each instance of Raa is, independently, selected from C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Raa groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;
[0339] each instance of Rbb is, independently, selected from hydrogen, -OH, -0Raa, - N(Rcc)2, -CN, -C(=0)Raa, -C(=0)N(Rcc)2, -CO2Raa, -SO2Raa, -C(=NRcc)0Raa, -C(=NRcc)N(Rcc)2, -SO2N(Rcc)2, -SO2Rcc, -S020Rcc, -SORaa, -C(=S)N(Rcc)2, -C(=0)SRcc, - C(=S)SRcc, -P(=0)2Raa, -P(=0)(Raa)2, -P(=0)2N(Rcc)2, -P(=0)(NRcc)2, C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Rbb groups, together with the heteroatom to which they are attached, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;
[0340] each instance of Rcc is, independently, selected from hydrogen, C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Rcc groups, together with the heteroatom to which they are attached, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;

[0341] each instance of Rdd is, independently, selected from halogen, -CN, -NO2, -N3, - SO2H, -S03H, -OH, -0Ree, -0N(Rff)2, -N(Rff)2, -N(Rff)3+X-, -N(ORee)Rff, -SH, -SRee, -SSRee, -C(=0)Ree, -CO2H, -CO2Ree, -0C(=0)Ree, -0CO2Ree, -C(=0)N(Rff)2, - OC(=0)N(Rff)2, -NRffC(=0)Ree, -NRffCO2Ree, -NRffC(=0)N(Rff)2, -C(=NRff)0Ree, - OC(=NRff)Ree, -0C(=NRMORee, -C(=NRff)N(Rff)2, -0C(=NRff)N(Rff)2, -NRffC(=NRff)N(Rff)2, -NRffS02Ree, -SO2N(Rff)2, -SO2Ree, -S020Ree, -0S02Ree, -S(=0)Ree, -Si(Ree)3, -0Si(Ree)3, -C(=S)N(Rff)2, -C(=0)SRee, -C(=S)SRee, -SC(=S)SRee, -P(=0)2Ree, - P(=0)(Ree)2, -0P(=0)(Ree)2, -OP(=0)(0Ree)2, C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, 3-membered heterocyclyl, C6-10 aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups, or two geminal Rdd substituents can be joined to form =0 or =S;
[0342] each instance of Ree is, independently, selected from C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, C6-10 aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups;
[0343] each instance of Rff is, independently, selected from hydrogen, C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, 3-10 membered heterocyclyl, C6-10 aryl and 5-10 membered heteroaryl, or two Rff groups, together with the heteroatom to which they are attached, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups; and [0344] each instance of Rgg is, independently, halogen, -CN, -NO2, -N3, -S02H, -S03H, -OH, -0C1-50 alkyl, -0N(C1-50 alky1)2, -N(C1-50 alky1)2, -N(C1-50 alky1)3+X-, -NH(C1-50 alky1)2+X-, -NH2(C1-50 alkyl) +X-, -NH3+X-, -N(0C1-50 alkyl)(C1-50 alkyl), -N(OH)(C1-50 alkyl), -NH(OH), -SH, -SC1-50 alkyl, -SS(C1-50 alkyl), -C(=0)(C1-50 alkyl), -CO2H, -CO2(C1-50 alkyl), -0C(=0)(C1-50 alkyl), -00O2(C1-50 alkyl), -C(=0)NH2, -C(=0)N(C1-50 alky1)2, -OC(=0)NH(C1-50 alkyl), -NHC(=0)(C1-50 alkyl), -N(C1-50 alkyl)C(=0)(C1-50 alkyl), -NHCO2(C1-50 alkyl), -NHC(=0)N(C1-50 alky1)2, -NHC(=0)NH(C1-50 alkyl), - NHC(=0)NH2, -C(NH)0(C1-5O alkyl),-0C(=NH)(C1-50 alkyl), -0C(NH)0C1-5O alkyl, - C(=NH)N(C1-50 alky1)2, -C(=NH)NH(C1-50 alkyl), -C(=NH)NH2, -0C(=NH)N(C1-50alky1)2, -0C(NH)NH(C1-50 alkyl), -OC(NH)NH2, -NHC(NH)N(C1-50 alky1)2, - NHC(=NH)NH2, -NHS02 (C1-50 alkyl), -S
02N (C1-50 alky1)2, -SO2NH (C 1-50 alkyl), - SO2NH2,-S02C1-50 alkyl, -S020C1-50 alkyl, -0S02C1-6 alkyl, -SOC1-6 alkyl, -Si(C1-50 alky1)3, -0Si(C1-6 alky1)3 -C(=S)N(C1-50 alky1)2, C(=S)NH(C1-50 alkyl), C(=S)NH2, - C(=0)S(C1-6 alkyl), -C(=S)SC1-6 alkyl, -SC(=S)SC1-6 alkyl, -P(=0)2(C1-50 alkyl), -P(=0)(C1-50 alky1)2, -0P(=0)(C1-50 alky1)2, -0P(=0)(0C1-50 alky1)2, C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, C6-10 aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl;
or two geminal Rgg substituents can be joined to form =0 or =S;
wherein X- is a counterion.
[0345] As used herein, the term "halo" or "halogen" refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).
[0346] As used herein, a "counterion" is a negatively charged group associated with a positively charged quarternary amine in order to maintain electronic neutrality. Exemplary counterions include halide ions (e.g., F-, Cl-, Br-, I-), NO3-, C104-, OH-, H2PO4-, HSO4-, sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-l-sulfonic acid-5-sulfonate, ethan-l-sulfonic acid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate, ethanoate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, and the like).
[0347] Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quarternary nitrogen atoms. Exemplary nitrogen atom substitutents include, but are not limited to, hydrogen, -OH, -0Raa, -N(Rcc)2, -CN, - C(=0)Raa, -C(=0)N(Rec)2, -CO2Raa, -SO2Raa, -C(=NRbb)Raa, -C(=NRcc)0Raa, -C(=NRcc)N(Rec)2, -SO2N(Rcc)2, -SO2Rcc, -S020Rcc, -SORaa, -C(=S)N(Rec)2, -C(=0)SRec, - C(=S)SRec, -P(=0)2Raa, -P(=0)(Raa)2, -P(=0)2N(Rec)2, -P(=0)(NRcc)2, C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Rcc groups, together with the N atom to which they are attached, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups, and wherein Raa, Rbb, Rcc and Rdd are as defined above.
[0348] Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quarternary nitrogen atoms. Exemplary nitrogen atom substitutents include, but are not limited to, hydrogen, -OH, -0Raa, -N(Rcc)2, -CN, - C(=0)Raa, -C(=0)N(Rec)2, -CO2Raa, -SO2Raa, -C(=NRbb)Raa, -C(=NRcc)0Raa, -C(=NRcc)N(Rec)2, -SO2N(Rcc)2, -SO2Rcc, -S020Rcc, -SORaa, -C(=S)N(Rcc)2, -C(=0)SRcc, - C(=S)SRcc, -P(=0)2Raa, -P(=0)(Raa)2, -P(=0)2N(Rcc)2, -P(=0)(NRcc)2, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Rcc groups, together with the nitrogen atom to which they are attached, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups, and wherein Raa, Rbb, Rcc and Rdd are as defined above.
[0349] In certain embodiments, the substituent present on a nitrogen atom is a nitrogen protecting group (also referred to as an amino protecting group). Nitrogen protecting groups include, but are not limited to, -OH, -0Raa, -N(Rcc)2, -C(=0)Raa, -C(=0)N(Rcc)2, -CO2Raa, -SO2Raa, -C(=NRcc)Raa, -C(=NRcc)0Raa, -C(=NRcc)N(Rcc)2, -SO2N(Rcc)2, -SO2Rcc, -S020Rcc, -SORaa, -C(=S)N(Rcc)2, -C(=0)SRcc, -C(=S)SRcc, C1-10 alkyl (e.g., aralkyl, heteroaralkyl), C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 Rdd groups, and wherein Raa, Rbb, Rcc and Rdd are as defined herein.
Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
[0350] For example, nitrogen protecting groups such as amide groups (e.g., - C(=0)Raa) include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methy1-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methy1-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

[0351] Nitrogen protecting groups such as carbamate groups (e.g., -C(=0)0Raa) include, but are not limited to, methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1- (1-adamanty1)-1-methylethyl carbamate (Adpoc), 1,1-dimethy1-2-haloethyl carbamate, 1,1-dimethy1-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethy1-2,2,2-trichloroethyl carbamate (TCBOC), 1-methy1-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylpheny1)-1-methylethyl carbamate (t-Bumeoc), 2-(2'-and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylally1 carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinoly1 carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4- methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethy1-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethy1-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-l-cyclopropylmethyl carbamate, 1-methy1-1(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-l-phenylethyl carbamate, 1- methyl-1-(4-pyridypethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.
[0352] Nitrogen protecting groups such as sulfonamide groups (e.g., -S(=0)2Raa) include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethy1-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethy1-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethy1-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6- trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), 13-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.
[0353] Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivative, N'-p-toluenesulfonylaminoacyl derivative, N' -phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-dipheny1-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4- tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethy1-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzy1-1,3,5-triazacyclohexan-2-one, 1- substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N42-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropy1-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7 -dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N' ,N'-dimethylaminomethylene)amine, N,N' -isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5- chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethy1-3-oxo-l-cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4- dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).
[0354] In certain embodiments, the substituent present on an oxygen atom is an oxygen protecting group (also referred to as a hydroxyl protecting group). Oxygen protecting groups include, but are not limited to, -Raa, -N(Rbb)2, -C(=0)SRaa, -C(=0)Raa, -CO2Raa, -C(=0)N(Rbb)2, -C(=NRbb)Raa, -C (=NRbb)0Raa, -C(=NRbb)N(Rbb)2, -S(=0)Raa, -SO2Raa, Si(Raa)3, -P(Rcc)2, -P(Rcc)3, -P(=0)2Raa, -P(=0)(Raa)2, -P(=0)(ORcc)2, -P(=0)2N(Rbb)2, and -P(=0)(NRbb)2, wherein Raa, Rbb, and Rcc are as defined herein. Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley &
Sons, 1999, incorporated herein by reference.
[0355] Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4- methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)pheny1]-4-methoxypiperidin-4-y1 (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethy1-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-l-methoxyethyl, 1-methyl-l-benzyloxyethyl, 1-methyl-l-b enzyloxy-2-fluoro ethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2- (phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3- methyl-2-picoly1 N-oxido, diphenylmethyl, p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4,4',4"-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4"-dimethoxyphenyl)methyl, 1,1-bis(4-methoxypheny1)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsily1 (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-l-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, (methylthiomethoxy)butyrate, 2- (methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methy1-2-butenoate, o-(methoxyacyl)benzoate, a-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).
[0356] In certain embodiments, the substituent present on an sulfur atom is an sulfur protecting group (also referred to as a thiol protecting group). Sulfur protecting groups include, but are not limited to, -Raa, -N(Rbb)2, -C(=0)SRaa, -C(=0)Raa, -CO2Raa, C(=0)N(Rbb)2, -C(=NRbb)Raa, -C(=NRbb)0Raa, -C(=NRbb)N(Rbb)2, -S(=0)Raa, -SO2Raa, -Si(Raa)3, -P(Rcc)2, -P(Rcc)3, -P(=0)2Raa, -P(=0)(Raa)2, -P(=0)(ORcc)2, -P(=0)2N(Rbb)2, and -P(=0)(NRbb)2, wherein Raa, Rbb, and Rcc are as defined herein. Sulfur protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
[0357] As used herein, a "leaving group" is an art-understood term referring to a molecular fragment that departs with a pair of electrons in heterolytic bond cleavage, wherein the molecular fragment is an anion or neutral molecule. See, for example, Smith, March's Advanced Organic Chemistry 6th ed. (501-502). Exemplary leaving groups include, but are not limited to, halo (e.g., chloro, bromo, iodo) and sulfonyl substituted hydroxyl groups (e.g., tosyl, mesyl, besyl).
Other definitions [0358] As used herein, use of the phrase "at least one instance" refers to one instance, but also encompasses more than one instance, e.g., for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 instances, and up to 100 instances.
[0359] As used herein, a "polymer" refers to a compound comprised of at least 3 (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.) repeating covalently bound structural units.
[0360] "Attached" refers to the covalent attachment of a group.
[0361] As used herein, "lipophilic" refers to the ability of a group to dissolve in fats, oils, lipids, and lipophilic non-polar solvents such as hexane or toluene. In general, a lipophilic group refers to an unsubstituted n-alkyl or unsubstituted n-alkenyl group having 6 to 50 carbon atoms, e.g., 6 to 40, 6 to 30, 6 to 20, 8 to 20, 8 to 19, 8 to 18, 8 to 17, 8 to 16, or 8 to 15 carbon atoms.

[0362] As used herein, the term "salt" or "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19.
Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or rnalonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate. digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4alky1)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium. quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate. Further pharmaceutically acceptable salts include salts formed from the quarternization of an amine using an appropriate electrophile, e.g., an alkyl halide, to form a quarternized alkylated amino salt.
Second or Additional Cationic Lipids [0363] In some embodiments, liposomes may comprise a second or additional cationic lipid. As used herein, the phrase "cationic lipid" refers to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available. Particularly suitable cationic lipids for use in the compositions and methods of the invention include those described in international patent publications WO 2010/053572 (and particularly, C12-200 described at paragraph [00225]) and WO 2012/170930, both of which are incorporated herein by reference. In certain embodiments, the compositions and methods of the invention employ a lipid nanoparticles comprising an ionizable cationic lipid described in U.S.
provisional patent application 61/617,468, filed March 29, 2012 (incorporated herein by reference), such as, e.g, (15Z, 18Z)-N,N-dimethy1-6-(9Z, 12Z)-octadeca-9, 12-dien-1 -yl)tetracosa- 15,18-dien- 1 -amine (HGT5000), ( 15Z, 18Z)-N,N-dimethy1-6-((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)tetracosa-4,15,18-trien-1 -amine (HGT5001), and (15Z,18Z)-N,N-dimethy1-64(9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)tetracosa-5, 15 , 18-trien- 1 -amine (HGT5002).
[0364] In some embodiments, the second or additional cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or "DOTMA" is used.
(Feigner et al.
(Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355). DOTMA can be formulated alone or can be combined with the neutral lipid, dioleoylphosphatidyl-ethanolamine or "DOPE"
or other cationic or non-cationic lipids into a liposomal transfer vehicle or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of nucleic acids into target cells. Other suitable cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide or "DOGS," 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propanaminium or "DOSPA" (Behr et al. Proc. Nat.'1 Acad. Sci. 86, 6982 (1989); U.S. Pat. No.
5,171,678; U.S. Pat.
No. 5,334,761), 1,2-Dioleoy1-3-Dimethylammonium-Propane or "DODAP", 1,2-Dioleoy1-3-Trimethylammonium-Propane or "DOTAP". Additional exemplary cationic lipids also include 1,2-distearyloxy-N,N-dimethy1-3-aminopropane or "DSDMA", 1,2-dioleyloxy-N,N-dimethy1-3-aminopropane or "DODMA", 1 ,2-dilinoleyloxy-N,N-dimethy1-3-aminopropane or "DLinDMA", 1,2-dilinolenyloxy-N,N-dimethy1-3-aminopropane or "DLenDMA", N-dioleyl-N,N-dimethylammonium chloride or "DODAC", N,N-distearyl-N,N-dimethylarnrnonium bromide or "DDAB", N-(1,2-dimyristyloxyprop-3-y1)-N,N-dimethyl-N-hydroxyethyl ammonium bromide or "DMRIE", 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(ci s,cis-9,12-octadecadienoxy)propane or "CLinDMA", 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethy 1-1-(cis,cis-9', 1-2'-octadecadienoxy)propane or "CpLinDMA", N,N-dimethy1-3,4-dioleyloxybenzylamine or "DMOBA", 1 ,2-N,N'-dioleylcarbamy1-3-dimethylaminopropane or "DOcarbDAP", 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or "DLinDAP", 1,2-N,N'-Dilinoleylcarbamy1-3-dimethylaminopropane or "DLincarbDAP", 1 ,2-Dilinoleoylcarbamy1-3-dimethylaminopropane or "DLinCDAP", 2,2-dilinoley1-4-dimethylaminomethyl-[1,3]-dioxolane or "DLin- -DMA", 2,2-dilinoley1-4-dimethylaminoethyl-[1,3]-dioxolane or "DLin-DMA", and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien- 1-y1)-1 ,3-dioxolan-4-y1)-N,N-dimethylethanamine (DLin-KC2-DMA)) (See, WO 2010/042877; Semple et al., Nature Biotech.
28: 172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, DV., et al., Nat. Biotechnol. 23(8): 1003-1007 (2005); PCT
Publication W02005/121348A1). In some embodiments, one or more of the cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.
[0365] In some embodiments, the second or additional cationic lipid may be chosen from XTC (2,2-Dilinoley1-4-dimethylaminoethyl-[1,3]-dioxolane), MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-y1 4-(dimethylamino)butanoate), ALNY-100 ((3aR,5s,6a5)-N,N-dimethy1-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d]
[1 ,3]dioxol-5-amine)), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propy1)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide), DODAP (1,2-dioley1-3-dimethylammonium propane), HGT4003 (WO 2012/170889, the teachings of which are incorporated herein by reference in their entirety), ICE (WO 2011/068810, the teachings of which are incorporated herein by reference in their entirety), HGT5000 (U.S. Provisional Patent Application No.
61/617,468, the teachings of which are incorporated herein by reference in their entirety) or HGT5001 (cis or trans) (Provisional Patent Application No. 61/617,468), aminoalcohol lipidoids such as those disclosed in W02010/053572, DOTAP (1,2-dioley1-3-trimethylammonium propane), DOTMA
(1,2-di-O-octadeceny1-3-trimethylammonium propane), DLinDMA (Heyes, J.;
Palmer, L.;
Bremner, K.; MacLachlan, I. "Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids" J. Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA
(Semple, S.C.
et al. "Rational Design of Cationic Lipids for siRNA Delivery" Nature Biotech.
2010, 28, 172-176), C12-200 (Love, K.T. et al. "Lipid-like materials for low-dose in vivo gene silencing"
PNAS 2010, 107, 1864-1869).
Non-cationic/Helper Lipids [0366] In some embodiments, provided liposomes contain one or more non-cationic ("helper") lipids. As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase "anionic lipid"
refers to any of a number of lipid species that carry a net negative charge at a selected H, such as physiological pH. Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-0-monomethyl PE, 16-0-dimethyl PE, 18-1-trans PE, 1-stearoy1-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixture thereof [0367] In some embodiments, such non-cationic lipids may be used alone, but are preferably used in combination with other excipients, for example, cationic lipids. In some embodiments, the non-cationic lipid may comprise a molar ratio of about 5% to about 90%, or about 10 % to about 70% of the total lipid present in a liposome. In some embodiments, a non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge in the conditions under which the composition is formulated and/or administered. In some embodiments, the percentage of non-cationic lipid in a liposome may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.
Cholesterol-based Lipids [0368] In some embodiments, provided liposomes comprise one or more cholesterol-based lipids. For example, suitable cholesterol-based cationic lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al.
BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or ICE. In some embodiments, the cholesterol-based lipid may comprise a molar ration of about 2% to about 30%, or about 5% to about 20% of the total lipid present in a liposome. In some embodiments, The percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than 5, %, 10%, greater than 20%, greater than 30%, or greater than 40%.
PEGylated Lipids [0369] In some embodiments, provided liposomes comprise one or more PEGylated lipids. For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide) is also contemplated by the present invention in combination with one or more of the cationic and, in some embodiments, other lipids together which comprise the liposome.
Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. In some embodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target cell, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613).
[0370] In some embodiments, particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18). The PEG-modified phospholipid and derivitized lipids of the present invention may comprise a molar ratio from about 0% to about 15%, about 0.5% to about 15%, about 1% to about 15%, about 4% to about 10%, or about 2%
of the total lipid present in the liposome.
[0371] According to various embodiments, the selection of second or additional cationic lipids, non-cationic lipids and/or PEG-modified lipids which comprise the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the mRNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus the molar ratios may be adjusted accordingly. In some embodiments, the percentage of PEG-modified lipid in a liposome may be greater than 1%, greater than 2%, greater than 5%, greater than 10%, or greater than 15%.
Polymer [0372] In some embodiments, a suitable liposome according to the present invention further includes a polymer, in combination with one or more cationic lipids as described and, in some embodiments, other carriers including various lipids described herein.
Thus, in some embodiments, liposomal delivery vehicles, as used herein, also encompass polymer containing nanoparticles. Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL, PEGylated PLL and polyethylenimine (PEI). When PEI is present, it may be branched PEI of a molecular weight ranging from 10 to 40 kDA, e.g., 25 kDa branched PEI
(Sigma #408727).
[0373] In some embodiments, a suitable liposome formulation contains a combination of one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids one or more PEG-modified lipids, and/or one or more polymers. As a non-limiting example, a suitable liposome comprises cKK-E12, DOPE, cholesterol and DMG-PEG2K. In some embodiments, the ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEGylated lipid may be between about 30-50:25-35:20-30:1-15, respectively. In some embodiments, the ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEGylated lipid is approximately 40:30:20:10, respectively. In some embodiments, the ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEGylated lipid is approximately 40:30:25:5, respectively. In some embodiments, the ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEGylated lipid is approximately 40:32:25:3, respectively.
mRNA
[0374] The present invention can be used to deliver any mRNA. mRNA is typically thought of as the type of RNA that carries information from DNA to the ribosome. The existence of mRNA is usually very brief and includes processing and translation, followed by degradation. Typically, in eukaryotic organisms, mRNA processing comprises the addition of a "cap" on the N-terminal (5') end, and a "tail" on the C-terminal (3') end. A
typical cap is a 7-methylguanosine cap, which is a guanosine that is linked through a 5'-5'-triphosphate bond to the first transcribed nucleotide. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The tail is typically a polyadenylation event whereby a polyadenylyl moiety is added to the 3' end of the mRNA molecule. The presence of this "tail"
serves to protect the mRNA from exonuclease degradation. Messenger RNA
typically is translated by the ribosomes into a series of amino acids that make up a protein.
[0375] Any mRNA capable of being translated into one or more peptides (e.g., proteins) or peptide fragments is contemplated as within the scope of the present invention. In some embodiments, an mRNA encodes one or more naturally occurring peptides. In some embodiments, an mRNA encodes one or more modified or non-natural peptides.
[0376] In some embodiments an mRNA encodes an intracellular protein. In some embodiments, an mRNA encodes a cytosolic protein. In some embodiments, an mRNA
encodes a protein associated with the actin cytoskeleton. In some embodiments, an mRNA
encodes a protein associated with the plasma membrane. In some specific embodiments, an mRNA
encodes a transmembrane protein. In some specific embodiments an mRNA encodes an ion channel protein. In some embodiments, an mRNA encodes a perinuclear protein.
In some embodiments, an mRNA encodes a nuclear protein. In some specific embodiments, an mRNA
encodes a transcription factor. In some embodiments, an mRNA encodes a chaperone protein.
In some embodiments, an mRNA encodes an intracellular enzyme (e.g., mRNA
encoding an enzyme associated with urea cycle or lysosomal storage metabolic disorders).
In some embodiments, an mRNA encodes a protein involved in cellular metabolism, DNA
repair, transcription and/or translation. In some embodiments, an mRNA encodes an extracellular protein. In some embodiments, an mRNA encodes a protein associated with the extracellular matrix. In some embodiments an mRNA encodes a secreted protein. In specific embodiments, an mRNA used in the composition and methods of the invention may be used to express functional proteins or enzymes that are excreted or secreted by one or more target cells into the surrounding extracellular fluid (e.g., mRNA encoding hormones and/or neurotransmitters).
[0377] In some embodiments, the compositions and methods of the invention provide for delivery of mRNA encoding a secreted protein. In some embodiments, the compositions and methods of the invention provide for delivery of mRNA encoding one or more secreted proteins listed in Table 1; thus, compositions of the invention may comprise an mRNA
encoding a protein listed in Table 1 (or a homolog thereof) along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a protein listed in Table 1 (or a homolog thereof) along with other components set out herein.

Table 1. Secreted Proteins Uniprot ID Protein Name Gene Name Al E959 Odontogenic ameloblast-associated protein ODAM
Al KZ92 Peroxidasin-like protein PXDNL
A1L453 Serine protease 38 PRSS38 Soluble scavenger receptor cysteine-rich domain-AlL4H1 SSC5D
containing protein S SC5D
A2RUU4 Colipase-like protein 1 CLPSL1 A2VDFO Fucose mutarotase FUOM
A2VEC9 SCO-spondin SSPO
von Willebrand factor A domain-containing protein 8 A4DOS4 Laminin subunit beta-4 LAMB4 A4D1T9 Probable inactive serine protease 37 PR5537 A5D8T8 C-type lectin domain family 18 member A CLEC18A
phospholipase A2 inhibitor and Ly6/PLAUR

domain-containing protein von Willebrand factor A domain-containing protein 3A
A6NDO1 Probable folate receptor delta FOLR4 A6NDD2 Beta-defensin 108B-like A6NE02 BTB/POZ domain-containing protein 17 BTBD17 A6NEF6 Growth hormone 1 GH1 A6NFO2 NPIP-like protein LOC730153 A6NFB4 HCG1749481, isoform CRA_k CSH1 A6NFZ4 Protein FAM24A FAM24A
A6NG13 Glycosyltransferase 54 domain-containing protein A6NGN9 IgLON family member 5 IGLON5 A6NHNO Otolin-1 OTOL1 A6NHN6 Nuclear pore complex-interacting protein-like 2 NPIPL2 Leukocyte immunoglobulin-like receptor subfamily A member 5 Chorionic somatomammotropin hormone 2 isoform 2 A6NJ69 IgA-inducing protein homolog IGIP
A6NKQ9 Choriogonadotropin subunit beta variant 1 CGB1 A6NMZ7 Collagen alpha-6(VI) chain COL6A6 A6NNS2 Dehydrogenase/reductase SDR family member 7C DHRS7C
A6XGL2 Insulin A chain INS
A8K0G1 Protein Wnt WNT7B
A8K2U0 Alpha-2-macroglobulin-like protein 1 A2ML1
133 A8K7I4 Calcium-activated chloride channel regulator 1 CLCA1 A8MTL9 Serpin-like protein HMSD HMSD
A8MV23 Serpin E3 SERPINE3 A8MZH6 Oocyte-secreted protein 1 homolog 00SP1 A8TX70 Collagen alpha-5(VI) chain COL6A5 BOZBE8 Natriuretic peptide NPPA
BlA4G9 Somatotropin GH1 B1A4H2 HCG1749481, isoform CRA_d CSH1 BlA4H9 Chorionic somatomammotropin hormone CSH2 B 1 AJZ6 Protein Wnt WNT4 B1AKI9 Isthmin-1 ISM1 Complement Clq and tumor necrosis factor-related protein 9B
von Willebrand factor C domain-containing protein 2-like B3GLJ2 Prostate and testis expressed protein 3 PATE3 B4DI03 SEC11-like 3 (S. cerevisiae), isoform CRA_a SEC11L3 B4DJF9 Protein Wnt WNT4 B4DUL4 SEC11-like 1(S. cerevisiae), isoform CRA_d SEC11L1 B5MCC8 Protein Wnt WNT1OB
B8A595 Protein Wnt WNT7B
B8A597 Protein Wnt WNT7B
B8A598 Protein Wnt WNT7B
B9A064 Immunoglobulin lambda-like polypeptide 5 IGLL5 C9J3H3 Protein Wnt WNT1OB
C9J8I8 Protein Wnt WNT5A
C9JAF2 Insulin-like growth factor II Ala-25 Del IGF2 C9JCI2 Protein Wnt WNT1OB
C9JL84 HERV-H LTR-associating protein 1 HHLA1 C9JNR5 Insulin A chain INS
C9JUI2 Protein Wnt WNT2 D6RF47 Protein Wnt WNT8A
D6RF94 Protein Wnt WNT8A
E2RYF7 Protein PBMUCL2 HCG22 E5RFR1 PENK(114-133) PENK
E7EML9 Serine protease 44 PR5544 E7EPC3 Protein Wnt WNT9B
E7EVP0 Nociceptin PNOC
E9PD02 Insulin-like growth factor I IGF1 E9PH60 Protein Wnt WNT16
134 E9PJL6 Protein Wnt WNT11 F5GYM2 Protein Wnt WNT5B
F5H034 Protein Wnt WNT5B
F5H364 Protein Wnt WNT5B
F5H7Q6 Protein Wnt WNT5B
F8WCM5 Protein INS-IGF2 INS-IGF2 F8WDR1 Protein Wnt WNT2 H0Y663 Protein Wnt WNT4 Signal peptidase complex catalytic subunit SEC 1 lA
Signal peptidase complex catalytic subunit SEC 1 lA
HOYM39 Chorionic somatomammotropin hormone CSH2 HOYMT7 Chorionic somatomammotropin hormone CSH1 HOYN1 7 Chorionic somatomammotropin hormone CSH2 Signal peptidase complex catalytic subunit SEC 1 lA
Signal peptidase complex catalytic subunit SEC 1 lA
Signal peptidase complex catalytic subunit SEC 1 lA
H7BZB8 Protein Wnt WNT 1 OA
H9KV56 Choriogonadotropin subunit beta variant 2 CGB2 I3L0L8 Protein Wnt WNT9B
J3KNZ1 Choriogonadotropin subunit beta variant 1 CGB1 J3KPOO Choriogonadotropin subunit beta CGB7 J3QT02 Choriogonadotropin subunit beta variant 1 CGB1 000175 C-C motif chemokine 24 CCL24 000182 Galectin-9 LGALS9 000187 Mannan-binding lectin serine protease 2 MASP2 000230 Cortistatin CORT
000253 Agouti-related protein AGRP
12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid receptor 000292 Left-right determination factor 2 LEFTY2 000294 Tubby-related protein 1 TULP 1 000295 Tubby-related protein 2 TULP2 Tumor necrosis factor receptor superfamily member 11B
000339 Matrilin-2 MATN2 000391 Sulfhydryl oxidase 1 QS0X1 000468 Agrin AGRN
000515 Ladinin-1 LAD 1
135 Processed neural cell adhesion molecule Li-like 000533 CHL1 protein 000584 Ribonuclease T2 RNASET2 000585 C-C motif chemokine 21 CCL21 000602 Ficolin-1 FCN1 000622 Protein CYR61 CYR61 000626 MDC(5-69) CCL22 000634 Netrin-3 NTN3 000744 Protein Wnt-10b WNT1OB
000755 Protein Wnt-7a WNT7A
Immunoglobulin superfamily containing leucine-rich repeat protein 014511 Pro-neuregulin-2, membrane-bound isoform NRG2 014594 Neurocan core protein NCAN
014625 C-X-C motif chemokine 11 CXCL11 Ectonucleotide 014638 pyrophosphatase/phosphodiesterase family ENPP3 member 3 014656 Torsin-1A TOR1A
014657 Torsin-1B TOR1B
014786 Neuropilin-1 NRP1 Tumor necrosis factor ligand superfamily member 11, membrane form 014791 Apolipoprotein Li APOL1 014793 Growth/differentiation factor 8 MSTN
014904 Protein Wnt-9a WNT9A
014905 Protein Wnt-9b WNT9B
014944 Proepiregulin EREG
014960 Leukocyte cell-derived chemotaxin-2 LECT2 015018 Processed PDZ domain-containing protein 2 PDZD2 015041 Semaphorin-3E SEMA3E
A disintegrin and metalloproteinase with thrombospondin motifs 3 015123 Angiopoietin-2 ANGPT2 015130 Neuropeptide FF NPFF
015197 Ephrin type-B receptor 6 EPHB6 015230 Laminin subunit alpha-5 LAMAS
015232 Matrilin-3 MATN3 015240 Neuroendocrine regulatory peptide-1 VGF
015263 Beta-defensin 4A DEFB4A
015335 Chondroadherin CHAD
136 015393 Transmembrane protease serine 2 catalytic chain TMPRSS2 015444 C-C motif chemokine 25 CCL25 015467 C-C motif chemokine 16 CCL16 015496 Group 10 secretory phospholipase A2 PLA2G1 0 015520 Fibroblast growth factor 10 FGF10 015537 Retinoschisin RS1 043157 Plexin-B 1 PLXNB 1 Disintegrin and metalloproteinase domain-containing protein 12 043240 Kallikrein-10 KLK10 043278 Kunitz-type protease inhibitor 1 SPINT 1 043320 Fibroblast growth factor 16 FGF16 043323 Desert hedgehog protein C-product DHH
043405 Cochlin COCH
Tumor necrosis factor ligand superfamily member 12, membrane form 043555 Progonadoliberin-2 GNRH2 Tumor necrosis factor ligand superfamily member 14, soluble form 043692 Peptidase inhibitor 15 PI15 043699 Sialic acid-binding Ig-like lectin 6 SIGLEC6 043 820 Hyaluronidase-3 HYAL3 043 827 Angiopoietin-related protein 7 ANGPTL7 043852 Calumenin CALU
EGF-like repeat and discoidin I-like domain-containing protein 3 043866 CD5 antigen-like CD5L
043 897 Tolloid-like protein 1 TLL1 043915 Vascular endothelial growth factor D FIGF
043927 C-X-C motif chemokine 13 CXCL 13 060218 Aldo-keto reductase family 1 member B10 AKR1B10 060235 Transmembrane protease serine 11D TMPRSS11D
060258 Fibroblast growth factor 17 FGF17 060259 Kallikrein-8 KLK8 060383 Growth/differentiation factor 9 GDF9 060469 Down syndrome cell adhesion molecule DSCAM
060542 Persephin PSPN
060565 Gremlin-1 GREM 1 060575 Serine protease inhibitor Kazal-type 4 SPINK4 060676 Cystatin-8 CST8 060687 Sushi repeat-containing protein SRPX2 SRPX2
137 060844 Zymogen granule membrane protein 16 ZG1 6 060882 Matrix metalloproteinase-20 MMP20 060938 Keratocan KERA
075015 Low affinity immunoglobulin gamma Fe region receptor III-B
Disintegrin and metalloproteinase domain-containing protein 23 075093 Slit homolog 1 protein SLIT1 075094 Slit homolog 3 protein SLIT3 Multiple epidermal growth factor-like domains protein 6 A disintegrin and metalloproteinase with thrombospondin motifs 4 075200 Nuclear pore complex-interacting protein-like 1 NPIPL1 075339 Cartilage intermediate layer protein 1 Cl CILP
075354 Ectonucleoside triphosphate diphosphohydrolase 6 ENTPD6 075386 Tubby-related protein 3 TULP3 Deformed epidermal autoregulatory factor 1 homolog 075443 Alpha-tectorin TECTA
075445 Usherin USH2A
075462 Cytokine receptor-like factor 1 CRLF1 075487 Glypican-4 GPC4 075493 Carbonic anhydrase-related protein 11 CA1 1 075594 Peptidoglycan recognition protein 1 PGLYRP 1 075596 C-type lectin domain family 3 member A CLEC3A
075610 Left-right determination factor 1 LEFTY1 075629 Protein CREG1 CREG 1 075636 Ficolin-3 FCN3 075711 Scrapie-responsive protein 1 SCRG1 075715 Epididymal secretory glutathione peroxidase GPX5 075718 Cartilage-associated protein CRTAP
075829 Chondrosurfactant protein LECT1 075830 Serpin 12 SERPINI2 075882 Attractin ATRN
Tumor necrosis factor ligand superfamily member 075900 Matrix metalloproteinase-23 MMP23A
075951 Lysozyme-like protein 6 LYZL6 075973 C 1 q-related factor C1QL1 076038 Secretagogin SCGN
076061 Stanniocalcin-2 STC2
138 076076 WNT1-inducible-signaling pathway protein 2 WISP2 076093 Fibroblast growth factor 18 FGF18 076096 Cystatin-F CST7 094769 Extracellular matrix protein 2 ECM2 094813 Slit homolog 2 protein C-product SLIT2 094907 Dickkopf-related protein 1 DKK1 094919 Endonuclease domain-containing 1 protein ENDOD 1 094964 N-terminal form SOGA1 095025 Semaphorin-3D SEMA3D
095084 Serine protease 23 PR5523 095150 Tumor necrosis factor ligand superfamily member 095156 Neurexophilin-2 NXPH2 095157 Neurexophilin-3 NXPH3 095158 Neurexophilin-4 NXPH4 095388 WNT1-inducible-signaling pathway protein 1 WISP1 095389 WNT1-inducible-signaling pathway protein 3 WISP3 095390 Growth/differentiation factor 11 GDF11 095393 Bone morphogenetic protein 10 BMP10 095399 Urotensin-2 UTS2 Tumor necrosis factor receptor superfamily member 6B
095428 Papilin PAPLN
095445 Apolipoprotein M APOM
A disintegrin and metalloproteinase with thrombospondin motifs 2 095460 Matrilin-4 MATN4 095467 LHAL tetrapeptide GNAS
095631 Netrin-1 NTN1 095633 Follistatin-related protein 3 FSTL3 095711 Lymphocyte antigen 86 LY86 095715 C-X-C motif chemokine 14 CXCL 14 095750 Fibroblast growth factor 19 FGF 19 095760 Interleukin-33 IL33 095813 Cerberus CER1 095841 Angiopoietin-related protein 1 ANGPTL1 095897 Noelin-2 OLFM2 095925 Eppin EPPIN
095965 Integrin beta-like protein 1 ITGBL1 095967 EGF-containing fibulin-like extracellular matrix protein 2
139 095968 Secretoglobin family 1D member 1 SCGB1D1 095969 Secretoglobin family 1D member 2 SCGB1D2 095970 Leucine-rich glioma-inactivated protein 1 LGI1 095972 Bone morphogenetic protein 15 BMP15 095994 Anterior gradient protein 2 homolog AGR2 095998 Interleukin-18-binding protein IL18BP
096009 Napsin-A NAP SA
096014 Protein Wnt-11 WNT11 P00450 Ceruloplasmin CP
P00451 Factor Villa light chain F8 P00488 Coagulation factor XIII A chain F13A1 P00533 Epidermal growth factor receptor EGFR
P00709 Alpha-lactalbumin LALBA
P00734 Prothrombin F2 P00738 Haptoglobin beta chain HP
P00739 Haptoglobin-related protein HPR
P00740 Coagulation factor IXa heavy chain F9 P00742 Factor X heavy chain F10 P00746 Complement factor D CFD
P00747 Plasmin light chain B PLG
P00748 Coagulation factor XIIa light chain F12 Urokinase-type plasminogen activator long chain A
P00750 Tissue-type plasminogen activator PLAT
P00751 Complement factor B Ba fragment CFB
P00797 Renin REN
P00973 2'-5'-oligoadenylate synthase 1 OAS1 P00995 Pancreatic secretory trypsin inhibitor SPINK1 P01008 Antithrombin-III SERPINC1 P01009 Alpha-l-antitrypsin SERPINA1 P01011 Alpha-l-antichymotrypsin His-Pro-less SERPINA3 P01019 Angiotensin-1 AGT
P01023 Alpha-2-macroglobulin A2M
P01024 Acylation stimulating protein C3 P01031 Complement C5 beta chain C5 P01033 Metalloproteinase inhibitor 1 TIMP1 P01034 Cystatin-C CST3 P01036 Cystatin-S CST4 P01037 Cystatin-SN CST1
140 P01042 Kininogen-1 light chain KNG1 P01127 Platelet-derived growth factor subunit B PDGFB
P01135 Transforming growth factor alpha TGFA
P01137 Transforming growth factor beta-1 TGFB1 P01138 Beta-nerve growth factor NGF
P01148 Gonadoliberin-1 GNRH1 P01160 Atrial natriuretic factor NPPA
P01178 Oxytocin OXT
P01185 Vasopressin-neurophysin 2-copeptin AVP
P01189 Corticotropin POMC
P01210 PENK(237-258) PENK
P01213 Alpha-neoendorphin PDYN
P01215 Glycoprotein hormones alpha chain CGA
P01222 Thyrotropin subunit beta TSHB
P01225 Follitropin subunit beta FSHB
P01229 Lutropin subunit beta LHB
P01233 Choriogonadotropin subunit beta CGB8 P01236 Prolactin PRL
P01241 Somatotropin GH1 P01242 Growth hormone variant GH2 P01243 Chorionic somatomammotropin hormone CSH2 P01258 Katacalcin CALCA
P01266 Thyroglobulin TG
P01270 Parathyroid hormone PTH
P01275 Glucagon GCG
P01282 Intestinal peptide PHM-27 VIP
P01286 Somatoliberin GHRH
P01298 Pancreatic prohormone PPY
P01303 C-flanking peptide of NPY NPY
P01308 Insulin INS
P01344 Insulin-like growth factor II IGF2 P01350 Big gastrin GAST
P01374 Lymphotoxin-alpha LTA
P01375 C-domain 1 TNF
P01562 Interferon alpha-1/13 IFNA1 P01563 Interferon alpha-2 IFNA2 P01566 Interferon alpha-10 IFNA10 P01567 Interferon alpha-7 IFNA7 P01568 Interferon alpha-21 IFNA21
141 P01569 Interferon alpha-5 IFNA5 P01570 Interferon alpha-14 IFNA14 P01571 Interferon alpha-17 IFNA17 P01574 Interferon beta IFNB1 P01579 Interferon gamma IFNG
P01583 Interleukin-1 alpha IL lA
P01584 Interleukin-1 beta IL 1B
P01588 Erythropoietin EPO
P01591 Immunoglobulin J chain IGJ
P01732 T-cell surface glycoprotein CD8 alpha chain CD8A
P01833 Polymeric immunoglobulin receptor PIGR
P01857 Ig gamma-1 chain C region IGHG1 P01859 Ig gamma-2 chain C region IGHG2 P01860 Ig gamma-3 chain C region IGHG3 P01861 Ig gamma-4 chain C region IGHG4 P01871 Ig mu chain C region IGHM
P01880 Ig delta chain C region IGHD
P02452 Collagen alpha-1(I) chain COL1A1 P02458 Chondrocalcin COL2A1 P02461 Collagen alpha-1(III) chain COL3A1 P02462 Collagen alpha-1(IV) chain COL4A1 P02647 Apolipoprotein A-I AP0A1 P02649 Apolipoprotein E APOE
P02652 Apolipoprotein A-II AP0A2 P02654 Apolipoprotein C-I APOC1 P02655 Apolipoprotein C-II APOC2 P02656 Apolipoprotein C-III APOC3 P02671 Fibrinogen alpha chain FGA
P02675 Fibrinopeptide B FGB
P02679 Fibrinogen gamma chain FGG
P02741 C-reactive protein CRP
P02743 Serum amyloid P-component(1-203) APCS
P02745 Complement Clq subcomponent subunit A C 1 QA
P02746 Complement Clq subcomponent subunit B C 1 QB
P02747 Complement Clq subcomponent subunit C C1 QC
P02748 Complement component C9b C9 P02749 Beta-2-glycoprotein 1 APOH
P02750 Leucine-rich alpha-2-glycoprotein LRG1 P02751 Ugl-Y2 FN1
142 P02753 Retinol-binding protein 4 RBP4 P02760 Trypstatin AMBP
P02763 Alpha-l-acid glycoprotein 1 ORM1 P02765 Alpha-2-HS-glycoprotein chain A AHSG
P02766 Transthyretin TTR
P02768 Serum albumin ALB
P02771 Alpha-fetoprotein AFP
P02774 Vitamin D-binding protein GC
P02775 Connective tissue-activating peptide III PPBP
P02776 Platelet factor 4 PF4 P02778 CXCL10(1-73) CXCL10 P02786 Transferrin receptor protein 1 TFRC
P02787 Serotransferrin TF
P02788 Lactoferroxin-C LTF
P02790 Hemopexin HPX
P02808 Statherin STATH
P02810 Salivary acidic proline-rich phosphoprotein 1/2 PRH2 P02812 Basic salivary proline-rich protein 2 PRB2 P02814 Peptide DIA SMR3B
P02818 Osteocalcin BGLAP
P03950 Angiogenin ANG
P03951 Coagulation factor XIa heavy chain F 11 P03952 Plasma kallikrein KLKB1 P03956 27 kDa interstitial collagenase MMP1 P03971 Muellerian-inhibiting factor AMH
P03973 Antileukoproteinase SLPI
P04003 C4b-binding protein alpha chain C4BPA
P04004 Somatomedin-B VTN
P04054 Phospholipase A2 PLA2G1B
P04085 Platelet-derived growth factor subunit A PDGFA
P04090 Relaxin A chain RLN2 P04114 Apolipoprotein B-100 APOB
P04118 Colipase CLPS
Granulocyte-macrophage colony-stimulating factor P04155 Trefoil factor 1 TFF1 P04180 Phosphatidylcholine-sterol acyltransferase LCAT
P04196 Histidine-rich glycoprotein HRG
P04217 Alpha-1B-glycoprotein AlBG
143 P04275 von Willebrand antigen 2 VWF
P04278 Sex hormone-binding globulin SHBG
P04279 Alpha-inhibin-31 SEMG1 P04280 Basic salivary proline-rich protein 1 PRB1 P04628 Proto-oncogene Wnt-1 WNT1 P04745 Alpha-amylase 1 AMY1A
P04746 Pancreatic alpha-amylase AMY2A
P04808 Prorelaxin H1 RLN1 P05000 Interferon omega-1 IFNW1 P05013 Interferon alpha-6 IFNA6 P05014 Interferon alpha-4 IFNA4 P05015 Interferon alpha-16 IFNA16 P05019 Insulin-like growth factor I IGF 1 P05060 GAWK peptide CHGB
P05090 Apolipoprotein D APOD
P05109 Protein S100-A8 5100A8 P05111 Inhibin alpha chain INHA
P05112 Interleukin-4 IL4 P05113 Interleukin-5 IL5 P05120 Plasminogen activator inhibitor 2 SERPINB2 P05121 Plasminogen activator inhibitor 1 SERPINE1 P05154 Plasma serine protease inhibitor SERPINA5 P05155 Plasma protease Cl inhibitor SERPING1 P05156 Complement factor I heavy chain CFI
P05160 Coagulation factor XIII B chain F13B
P05161 Ubiquitin-like protein ISG15 ISG15 P05230 Fibroblast growth factor 1 FGF 1 P05231 Interleukin-6 IL6 P05305 Big endothelin-1 EDN1 P05408 C-terminal peptide SCG5 P05451 Lithostathine-l-alpha REG lA
P05452 Tetranectin CLEC3B
P05543 Thyroxine-binding globulin SERPINA7 P05814 Beta-casein CSN2 P05997 Collagen alpha-2(V) chain COL5A2 P06276 Cholinesterase BCHE
P06307 Cholecystokinin-12 CCK
P06396 Gelsolin GSN
P06681 Complement C2 C2
144 P06702 Protein S100-A9 S100A9 P06727 Apolipoprotein A-TV AP0A4 Low affinity immunoglobulin epsilon Fe receptor soluble form P06744 Glucose-6-phosphate isomerase GPI
P06850 Corticoliberin CRH
P06858 Lipoprotein lipase LPL
P06881 Calcitonin gene-related peptide 1 CALCA
P07093 Glia-derived nexin SERPINE2 P07098 Gastric triacylglycerol lipase LIPF
P07225 Vitamin K-dependent protein S PROS1 P07237 Protein disulfide-isomerase P4HB
P07288 Prostate-specific antigen KLK3 P07306 Asialoglycoprotein receptor 1 ASGR1 P07355 Annexin A2 ANXA2 P07357 Complement component C8 alpha chain C8A
P07358 Complement component C8 beta chain C8B
P07360 Complement component C8 gamma chain C8G
P07477 Alpha-trypsin chain 2 PRSS1 P07478 Trypsin-2 PRSS2 P07492 Neuromedin-C GRP
P07498 Kappa-casein CSN3 P07585 Decorin DCN
P07911 Uromodulin UMOD
P07942 Laminin subunit beta-1 LAMB1 P07988 Pulmonary surfactant-associated protein B SFTPB
P07998 Ribonuclease pancreatic RNASE1 P08118 Beta-microseminoprotein MSMB
P08123 Collagen alpha-2(I) chain COL1A2 P08185 Corticosteroid-binding globulin SERPINA6 P08217 Chymotrypsin-like elastase family member 2A CELA2A
P08218 Chymotrypsin-like elastase family member 2B CELA2B
P08253 72 kDa type W collagenase MMP2 P08254 Stromelysin-1 MMP3 P08294 Extracellular superoxide dismutase [Cu-Zn] 50D3 P08476 Inhibin beta A chain INHBA
P08493 Matrix Gla protein MGP
P08572 Collagen alpha-2(IV) chain COL4A2 P08581 Hepatocyte growth factor receptor MET
145 P08603 Complement factor H CFH
P08620 Fibroblast growth factor 4 FGF4 Low affinity immunoglobulin gamma Fe region receptor III-A
P08697 Alpha-2-antiplasmin SERPINF2 P08700 Interleukin-3 IL3 P08709 Coagulation factor VII F7 P08833 Insulin-like growth factor-binding protein 1 IGFBP1 P08887 Interleukin-6 receptor subunit alpha IL6R
P08949 Neuromedin-B-32 NMB
P08F94 Fibrocystin PKHD1 P09038 Fibroblast growth factor 2 FGF2 P09228 Cystatin-SA CST2 P09237 Matrilysin MMP7 P09238 Stromelysin-2 MMP10 P09341 Growth-regulated alpha protein CXCL1 P09382 Galectin-1 LGALS1 P09466 Glycodelin PAEP

P09529 Inhibin beta B chain INHBB
P09544 Protein Wnt-2 WNT2 P09603 Processed macrophage colony-stimulating factor 1 CSF1 P09681 Gastric inhibitory polypeptide GIP
P09683 Secretin SCT
P09919 Granulocyte colony-stimulating factor CSF3 P00091 FRAS1-related extracellular matrix protein 3 FREM3 POCOL4 C4d-A C4A
POCOL5 Complement C4-B alpha chain C4B
POCOP6 Neuropeptide S NPS
POC7L1 Serine protease inhibitor Kazal-type 8 SPINK8 Complement Clq and tumor necrosis factor-related protein 9A
POC8F1 Prostate and testis expressed protein 4 PATE4 POCGO1 Gastrokine-3 GKN3P
POCG36 Cryptic family protein 1B CFC1B
POCG37 Cryptic protein CFC1 POCJ68 Humanin-like protein 1 MTRNR2L1 POCJ69 Humanin-like protein 2 MTRNR2L2 POCJ70 Humanin-like protein 3 MTRNR2L3 POCJ71 Humanin-like protein 4 MTRNR2L4
146 POCJ72 Humanin-like protein 5 MTRNR2L5 POCJ73 Humanin-like protein 6 MTRNR2L6 POCJ74 Humanin-like protein 7 MTRNR2L7 POCJ75 Humanin-like protein 8 MTRNR2L8 POCJ76 Humanin-like protein 9 MTRNR2L9 POCJ77 Humanin-like protein 10 MTRNR2L10 PODJD7 Pepsin A-4 PGA4 PODJD8 Pepsin A-3 PGA3 PODJD9 Pepsin A-5 PGA5 PODJI8 Amyloid protein A SAA1 PODJI9 Serum amyloid A-2 protein SAA2 P10082 Peptide YY(3-36) PYY
P10092 Calcitonin gene-related peptide 2 CALCB
P10124 Serglycin SRGN
P10145 MDNCF-a IL8 P10147 MIP-1-alpha(4-69) CCL3 P10163 Peptide P-D PRB4 P10451 Osteopontin SPP1 P10599 Thioredoxin TXN
P10600 Transforming growth factor beta-3 TGFB3 P10643 Complement component C7 C7 P10645 Vasostatin-2 CHGA
P10646 Tissue factor pathway inhibitor TFPI
P10720 Platelet factor 4 variant(4-74) PF4V1 P10745 Retinol-binding protein 3 RBP3 P10767 Fibroblast growth factor 6 FGF6 P10909 Clusterin alpha chain CLU
P10912 Growth hormone receptor GHR
P10915 Hyaluronan and proteoglycan link protein 1 HAPLN1 P10966 T-cell surface glycoprotein CD8 beta chain CD8B
P10997 Islet amyloid polypeptide IAPP
P11047 Laminin subunit gamma-1 LAMC1 P11150 Hepatic triacylglycerol lipase LIPC
P11226 Mannose-binding protein C MBL2 P11464 Pregnancy-specific beta-l-glycoprotein 1 PSG1 P11465 Pregnancy-specific beta-l-glycoprotein 2 PSG2 P11487 Fibroblast growth factor 3 FGF3 P11597 Cholesteryl ester transfer protein CETP
P11684 Uteroglobin SCGB1A1
147 P11686 Pulmonary surfactant-associated protein C SFTPC
P12034 Fibroblast growth factor 5 FGF5 P12107 Collagen alpha-1(XI) chain COL11A1 P12109 Collagen alpha-1(VI) chain COL6A1 P12110 Collagen alpha-2(VI) chain COL6A2 P12111 Collagen alpha-3(VI) chain COL6A3 P12259 Coagulation factor V F5 P12272 PTHrP[1-36] PTHLH
P12273 Prolactin-inducible protein PIP
P12544 Granzyme A GZMA
P12643 Bone morphogenetic protein 2 BMP2 P12644 Bone morphogenetic protein 4 BMP4 P12645 Bone morphogenetic protein 3 BMP3 P12724 Eosinophil cationic protein RNASE3 P12821 Angiotensin-converting enzyme, soluble form ACE
P12838 Neutrophil defensin 4 DEFA4 P12872 Motilin MLN
P13232 Interleukin-7 IL7 P13236 C-C motif chemokine 4 CCL4 Gamma-interferon-inducible lysosomal thiol reductase P13500 C-C motif chemokine 2 CCL2 P13501 C-C motif chemokine 5 CCL5 P13521 Secretogranin-2 SCG2 P13591 Neural cell adhesion molecule 1 NCAM1 P13611 Versican core protein VCAN
P13671 Complement component C6 C6 Carcinoembryonic antigen-related cell adhesion molecule 1 P13725 Oncostatin-M OSM
P13726 Tissue factor F3 P13727 Eosinophil granule major basic protein PRG2 P13942 Collagen alpha-2(XI) chain COL11A2 P13987 CD59 glycoprotein CD59 P14138 Endothelin-3 EDN3 P14174 Macrophage migration inhibitory factor MIF
P14207 Folate receptor beta FOLR2 P14222 Perforin-1 PRF1 P14543 Nidogen-1 NID1 P14555 Phospholipase A2, membrane associated PLA2G2A
148 P14625 Endoplasmin HSP90B1 P14735 Insulin-degrading enzyme IDE
P14778 Interleukin-1 receptor type 1, soluble form IL1R1 P14780 82 kDa matrix metalloproteinase-9 MMP9 P15018 Leukemia inhibitory factor LIF
P15085 Carboxypeptidase Al CPA1 P15086 Carboxypeptidase B CPB1 P15151 Poliovirus receptor PVR
P15169 Carboxypeptidase N catalytic chain CPN1 P15248 Interleukin-9 IL9 P15291 N-acetyllactosamine synthase B4GALT1 P15309 PAPf39 ACPP
P15328 Folate receptor alpha FOLR1 P15374 Ubiquitin carboxyl-terminal hydrolase isozyme L3 UCHL3 P15502 Elastin ELN
Granulocyte-macrophage colony-stimulating factor receptor subunit alpha P15515 Histatin-1 HTN1 P15516 His3-(31-51)-peptide HTN3 P15692 Vascular endothelial growth factor A VEGFA
P15814 Immunoglobulin lambda-like polypeptide 1 IGLL1 P15907 Beta-galactoside alpha-2,6-sialyltransferase 1 ST6GAL1 P15941 Mucin-1 subunit beta MUC1 P16035 Metalloproteinase inhibitor 2 TIMP2 P16112 Aggrecan core protein 2 ACAN
P16233 Pancreatic triacylglycerol lipase PNLIP
P16442 Histo-blood group ABO system transferase ABO
P16471 Prolactin receptor PRLR
P16562 Cysteine-rich secretory protein 2 CRISP2 P16619 C-C motif chemokine 3-like 1 CCL3L1 P16860 BNP(3-29) NPPB
P16870 Carboxypeptidase E CPE
P16871 Interleukin-7 receptor subunit alpha IL7R
P17213 Bactericidal permeability-increasing protein BPI
P17538 Chymotrypsinogen B CTRB1 P17931 Galectin-3 LGALS3 P17936 Insulin-like growth factor-binding protein 3 IGFBP3 P17948 Vascular endothelial growth factor receptor 1 FLT1 P18065 Insulin-like growth factor-binding protein 2 IGFBP2
149 P18075 Bone morphogenetic protein 7 BMP7 P18428 Lipopolysaccharide-binding protein LBP
P18509 PACAP-related peptide ADCYAP1 P18510 Interleukin-1 receptor antagonist protein IL1RN
P18827 Synde can-1 SDC1 Peptidylglycine alpha-hydroxylating monooxygenase P19235 Erythropoietin receptor EPOR
P19438 Tumor necrosis factor-binding protein 1 TNFRSF lA
P19652 Alpha-l-acid glycoprotein 2 ORM2 Amiloride-sensitive amine oxidase [copper-containing]
P19823 Inter-alpha-trypsin inhibitor heavy chain H2 ITIH2 P19827 Inter-alpha-trypsin inhibitor heavy chain H1 ITIH1 P19835 Bile salt-activated lipase CEL
P19875 C-X-C motif chemokine 2 CXCL2 P19876 C-X-C motif chemokine 3 CXCL3 P19883 Follistatin FST
P19957 Elafin PI3 P19961 Alpha-amylase 2B AMY2B
P20061 Transcobalamin-1 TCN1 P20062 Transcobalamin-2 TCN2 P20142 Gastricsin PGC
P20155 Serine protease inhibitor Kazal-type 2 SPINK2 P20231 Tryptase beta-2 TPSB2 Tumor necrosis factor receptor superfamily member 1B
P20366 Substance P TAC1 P20382 Melanin-concentrating hormone PMCH
P20396 Thyroliberin TRH
P20742 Pregnancy zone protein PZP
P20774 Mimecan OGN
P20783 Neurotrophin-3 NTF3 P20800 Endothelin-2 EDN2 P20809 Interleukin-11 IL11 P20827 Ephrin-Al EFNA1 P20849 Collagen alpha-1(IX) chain COL9A1 P20851 C4b-binding protein beta chain C4BPB
P20908 Collagen alpha-1(V) chain COL5A1 P21128 Poly(U)-specific endoribonuclease ENDOU
150 P21246 Pleiotrophin PTN
P21583 Kit ligand KITLG
P21741 Midkine MDK
P21754 Zona pellucida sperm-binding protein 3 ZP3 P21781 Fibroblast growth factor 7 FGF7 P21802 Fibroblast growth factor receptor 2 FGFR2 P21810 Biglycan BGN
P21815 Bone sialoprotein 2 IBSP
P21860 Receptor tyrosine-protein kinase erbB-3 ERBB3 P21941 Cartilage matrix protein MATN1 P22003 Bone morphogenetic protein 5 BMP5 P22004 Bone morphogenetic protein 6 BMP6 P22079 Lactoperoxidase LPO
P22105 Tenascin-X TNXB
P22301 Interleukin-10 IL10 P22303 Acetylcholinesterase ACHE
P22352 Glutathione peroxidase 3 GPX3 P22362 C-C motif chemokine 1 CCL1 P22455 Fibroblast growth factor receptor 4 FGFR4 P22466 Galanin message-associated peptide GAL
P22692 Insulin-like growth factor-binding protein 4 IGFBP4 P22749 Granulysin GNLY
P22792 Carboxypeptidase N subunit 2 CPN2 P22891 Vitamin K-dependent protein Z PROZ
P22894 Neutrophil collagenase MMP8 P23142 Fibulin-1 FBLN1 P23280 Carbonic anhydrase 6 CA6 P23352 Anosmin-1 KALI
P23435 Cerebellin-1 CBLN1 P23560 Brain-derived neurotrophic factor BDNF
P23582 C-type natriuretic peptide NPPC
P23946 Chymase CMA1 P24043 Laminin subunit alpha-2 LAMA2 P24071 Immunoglobulin alpha Fc receptor FCAR
P24347 Stromelysin-3 MMP11 P24387 Corticotropin-releasing factor-binding protein CRHBP
P24592 Insulin-like growth factor-binding protein 6 IGFBP6 P24593 Insulin-like growth factor-binding protein 5 IGFBP5 P24821 Tenascin TNC
151 P24855 Deoxyribonuclease-1 DNA SE1 P25067 Collagen alpha-2(VIII) chain COL8A2 P25311 Zinc-alpha-2-glycoprotein AZGP1 P25391 Laminin subunit alpha-1 LAMA1 Tumor necrosis factor receptor superfamily member 6 P25940 Collagen alpha-3(V) chain COL5A3 Tumor necrosis factor receptor superfamily member 5 P26022 Pentraxin-related protein PTX3 PTX3 P26927 Hepatocyte growth factor-like protein beta chain MST1 P27169 Serum paraoxonase/arylesterase 1 PON1 P27352 Gastric intrinsic factor GIF
P27487 Dipeptidyl peptidase 4 membrane form DPP4 P27539 Embryonic growth/differentiation factor 1 GDF1 P27658 Vastatin COL8A1 P27797 Cah-eticulin CALR
P27918 Properdin CFP
P28039 Acyloxyacyl hydrolase AOAH
P28300 Protein-lysine 6-oxidase LOX
P28325 Cystatin-D CST5 P28799 Granulin-1 GRN
P29122 Proprotein convertase subtilisin/kexin type 6 PCSK6 P29279 Connective tissue growth factor CTGF
P29320 Ephrin type-A receptor 3 EPHA3 P29400 Collagen alpha-5(IV) chain COL4A5 P29459 Interleukin-12 subunit alpha IL12A
P29460 Interleukin-12 subunit beta IL12B
P29508 Serpin B3 SERPINB3 P29622 Kallistatin SERPINA4 P29965 CD40 ligand, soluble form CD4OLG
P30990 Neurotensin/neuromedin N NTS
P31025 Lipocalin-1 LCN1 P31151 Protein S100-A7 5100A7 P31371 Fibroblast growth factor 9 FGF9 P31431 Syndecan-4 SDC4 P31947 14-3-3 protein sigma SFN
P32455 Interferon-induced guanylate-binding protein 1 GBP1 P32881 Interferon alpha-8 IFNA8 P34096 Ribonuclease 4 RNASE4
152 P34130 Neurotrophin-4 NTF4 P34820 Bone morphogenetic protein 8B BMP8B
P35030 Trypsin-3 PRSS3 P35052 Secreted glypican-1 GPC1 P35070 Betacellulin BTC
P35225 Interleukin-13 IL13 P35247 Pulmonary surfactant-associated protein D SFTPD

P35542 Serum amyloid A-4 protein SAA4 P35555 Fibrillin-1 FBN1 P35556 Fibrillin-2 FBN2 P35625 Metalloproteinase inhibitor 3 TIMP3 Insulin-like growth factor-binding protein complex acid labile subunit P35916 Vascular endothelial growth factor receptor 3 FLT4 P35968 Vascular endothelial growth factor receptor 2 KDR
P36222 Chitinase-3-like protein 1 CHI3L1 P36952 Serpin B5 SERPINB5 P36955 Pigment epithelium-derived factor SERPINF1 P36980 Complement factor H-related protein 2 CFHR2 P39059 Collagen alpha-1(XV) chain COL15A1 P39060 Collagen alpha-1(XVIII) chain COL18A1 P39877 Calcium-dependent phospholipase A2 PLA2G5 P39900 Macrophage metalloelastase MMP12 P39905 Glial cell line-derived neurotrophic factor GDNF
P40225 Thrombopoietin THPO
P40967 M-alpha PMEL
P41159 Leptin LEP
P41221 Protein Wnt-5a WNT5A
P41222 Prostaglandin-H2 D-isomerase PTGDS
P41271 Neuroblastoma suppressor of tumorigenicity 1 NBL1 P41439 Folate receptor gamma FOLR3 P42127 Agouti-signaling protein ASIP
P42702 Leukemia inhibitory factor receptor LIFR
P42830 ENA-78(9-78) CXCL5 P43026 Growth/differentiation factor 5 GDF5 P43251 Biotinidase BTD
P43652 Afamin AFM
P45452 Collagenase 3 MMP13
153 P47710 Casoxin-D CSN1S1 P47929 Galectin-7 LGALS7B
P47972 Neuronal pentraxin-2 NPTX2 P47989 Xanthine oxidase XDH
P47992 Lymphotactin XCL1 Tumor necrosis factor ligand superfamily member 6, membrane form P48052 Carboxypeptidase A2 CPA2 P48061 Stromal cell-derived factor 1 CXCL12 P48304 Lithostathine-l-beta REG1B
P48307 Tissue factor pathway inhibitor 2 TFPI2 P48357 Leptin receptor LEPR
P48594 Serpin B4 SERPINB4 P48645 Neuromedin-U-25 NMU
P48740 Mannan-binding lectin serine protease 1 MASP1 P48745 Protein NOV homolog NOV
P48960 CD97 antigen subunit beta CD97 P49223 Kunitz-type protease inhibitor 3 SPINT3 P49747 Cartilage oligomeric matrix protein COMP
P49763 Placenta growth factor PGF
P49765 Vascular endothelial growth factor B VEGFB
P49767 Vascular endothelial growth factor C VEGFC
P49771 Fms-related tyrosine kinase 3 ligand FLT3LG
P49862 Kallikrein-7 KLK7 P49863 Granzyme K GZMK
P49908 Selenoprotein P SEPP1 P49913 Antibacterial protein FALL-39 CAMP
P50607 Tubby protein homolog TUB
P51124 Granzyme M GZMM
P51512 Matrix metalloproteinase-16 MMP16 P51654 Glypican-3 GPC3 P51671 Eotaxin CCL11 P51884 Lumican LUM
P51888 Prolargin PRELP
P52798 Ephrin-A4 EFNA4 P52823 Stanniocalcin-1 STC1 P53420 Collagen alpha-4(IV) chain COL4A4 P53621 Coatomer subunit alpha COPA
P54108 Cysteine-rich secretory protein 3 CRISP3
154 P54315 Pancreatic lipase-related protein 1 PNLIPRP1 P54317 Pancreatic lipase-related protein 2 PNLIPRP2 P54793 Arylsulfatase F ARSF
P55000 Secreted Ly-6/uPAR-related protein 1 SLURP1 P55001 Microfibrillar- associated protein 2 MFAP2 P55056 Apolipoprotein C-IV APOC4 P55058 Phospholipid transfer protein PLTP
P55075 Fibroblast growth factor 8 FGF8 P55081 Microfibrillar-associated protein 1 MFAP1 P55083 Microfibril-associated glycoprotein 4 MFAP4 P55107 Bone morphogenetic protein 3B GDF10 Mesencephalic astrocyte-derived neurotrophic factor Pancreatic secretory granule membrane major glycoprotein GP2 P55268 Laminin subunit beta-2 LAMB2 P55773 CCL23(30-99) CCL23 P55774 C-C motif chemokine 18 CCL18 P55789 FAD-linked sulfhydryl oxidase ALR GFER
P56703 Proto-oncogene Wnt-3 WNT3 P56704 Protein Wnt-3a WNT3A
P56705 Protein Wnt-4 WNT4 P56706 Protein Wnt-7b WNT7B
P56730 Neurotrypsin PRSS12 P56851 Epididymal secretory protein E3-beta EDDM3B
P56975 Neuregulin-3 NRG3 P58062 Serine protease inhibitor Kazal-type 7 SPINK7 P58215 Lysyl oxidase homolog 3 LOXL3 P58294 Prokineticin-1 PROK1 P58335 Anthrax toxin receptor 2 ANTXR2 A disintegrin and metalloproteinase with thrombospondin motifs 12 P58417 Neurexophilin-1 NXPH1 P58499 Protein FAM3B FAM3B
A disintegrin and metalloproteinase with thrombospondin motifs 20 P59665 Neutrophil defensin 1 DEFA1B
P59666 Neutrophil defensin 3 DEFA3 P59796 Glutathione peroxidase 6 GPX6 P59826 BPI fold-containing family B member 3 BPIFB3 P59827 BPI fold-containing family B member 4 BPIFB4
155 P59861 Beta-defensin 131 DEFB131 P60022 Beta-defensin 1 DEFB1 P60153 Inactive ribonuclease-like protein 9 RNASE9 Complement Clq tumor necrosis factor-related protein 8 P60852 Zona pellucida sperm-binding protein 1 ZP1 P60985 Keratinocyte differentiation-associated protein KRTDAP
P61109 Kidney androgen-regulated protein KAP
P61278 Somatostatin-14 SST
P61366 Osteocrin OSTN
P61626 Lysozyme C LYZ
P61769 Beta-2-microglobulin B2M
P61812 Transforming growth factor beta-2 TGFB2 P61916 Epididymal secretory protein El NPC2 P62502 Epididymal-specific lipocalin-6 LCN6 P62937 Peptidyl-prolyl cis-trans isomerase A PPIA
P67809 Nuclease-sensitive element-binding protein 1 YBX1 Signal peptidase complex catalytic subunit P67812 SEC1 lA
SEC1 lA
P78310 Coxsackievirus and adenovirus receptor CXADR
P78333 Secreted glypican-5 GPC5 P78380 Oxidized low-density lipoprotein receptor 1 OLR1 P78423 Processed fractalkine CX3CL1 P78509 Reelin RELN
P78556 CCL20(2-70) CCL20 P80075 MCP-2(6-76) CCL8 P80098 C-C motif chemokine 7 CCL7 Phosphatidylinositol-glycan-specific phospholipase D
P80162 C-X-C motif chemokine 6 CXCL6 P80188 Neutrophil gelatinase-associated lipocalin LCN2 P80303 Nucleobindin-2 NUCB2 P80511 Calcitermin 5100Al2 P81172 Hepcidin-25 HAMP
P81277 Prolactin-releasing peptide PRLH
P81534 Beta-defensin 103 DEFB103A
P81605 Dermcidin DCD
P82279 Protein crumbs homolog 1 CRB1 P82987 ADAMTS-like protein 3 ADAMTSL3 P83105 Serine protease HTRA4 HTRA4
156 P83110 Serine protease HTRA3 HTRA3 P83859 Orexigenic neuropeptide QRFP QRFP
P98088 Mucin-5AC MUC5AC
P98095 Fibulin-2 FBLN2 Basement membrane-specific heparan sulfate proteoglycan core protein P98173 Protein FAM3A FAM3A
Q00604 Norrin NDP
Q00796 Sorbitol dehydrogenase SORD
Q00887 Pregnancy-specific beta-1 -glycoprotein 9 PSG9 Q00888 Pregnancy-specific beta-l-glycoprotein 4 PSG4 Q00889 Pregnancy-specific beta-l-glycoprotein 6 PSG6 Q01523 HD5(56-94) DEFA5 Q01524 Defensin-6 DEFA6 Q01955 Collagen alpha-3(IV) chain COL4A3 Q02297 Pro-neuregulin-1, membrane-bound isoform NRG1 Q02325 Plasminogen-like protein B PLGLB1 Q02383 Semenogelin-2 SEMG2 Q02388 Collagen alpha-1(VII) chain COL7A1 Q02505 Mucin-3A MUC3A
Q02509 Otoconin-90 0C90 Q02747 Guanylin GUCA2A
Q02763 Angiopoietin-1 receptor TEK
Q02817 Mucin-2 MUC2 Q02985 Complement factor H-related protein 3 CFHR3 Q03167 Transforming growth factor beta receptor type 3 TGFBR3 Q03403 Trefoil factor 2 TFF2 Q03405 Urokinase plasminogen activator surface receptor PLAUR
Q03591 Complement factor H-related protein 1 CFHR1 Q03692 Collagen alpha-1(X) chain COL10A1 Q04118 Basic salivary proline-rich protein 3 PRB3 Q04756 Hepatocyte growth factor activator short chain HGFAC
Q04900 Sialomucin core protein 24 CD164 Q05315 Eosinophil lysophospholipase CLC
Q05707 Collagen alpha-1(XIV) chain COL14A1 Q05996 Processed zona pellucida sperm-binding protein 2 ZP2 Q06033 Inter-alpha-trypsin inhibitor heavy chain H3 ITIH3 Q06141 Regenerating islet-derived protein 3-alpha REG3A
Q06828 Fibromodulin FMOD
157 Q07092 Collagen alpha-1(XVI) chain COL16A1 Q07325 C-X-C motif chemokine 9 CXCL9 Q07507 Dermatopontin DPT
Q075Z2 Binder of sperm protein homolog 1 BSPH1 Q07654 Trefoil factor 3 TFF3 Q07699 Sodium channel subunit beta-1 SCN1B
Q08345 Epithelial discoidin domain-containing receptor 1 DDR1 Q08380 Galectin-3-binding protein LGALS3BP
Q08397 Lysyl oxidase homolog 1 LOXL1 Q08431 Lactadherin MFGE8 Q08629 Testican-1 SPOCK1 Q08648 Sperm-associated antigen 11B SPAG11B
Q08830 Fibrinogen-like protein 1 FGL1 Q10471 Polypeptide N-acetylgalactosaminyltransferase 2 GALNT2 Q10472 Polypeptide N-acetylgalactosaminyltransferase 1 GALNT1 CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 1 CMP-N-acetylneuraminate-beta-1,4-galactoside alpha-2,3-sialyltransferase CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 4 Q12794 Hyaluronidase-1 HYAL1 EGF-containing fibulin-like extracellular matrix protein 1 Q12836 Zona pellucida sperm-binding protein 4 ZP4 Q12841 Follistatin-related protein 1 FSTL1 Aminoacyl tRNA synthase complex-interacting multifunctional protein 1 Q13018 Soluble secretory phospholipase A2 receptor PLA2R1 Q13072 B melanoma antigen 1 BAGE
Q13093 Platelet-activating factor acetylhydrolase PLA2G7 Q13103 Secreted phosphoprotein 24 SPP2 Q13162 Peroxiredoxin-4 PRDX4 Q13201 Platelet glycoprotein Ia* MMRN1 Q13214 Semaphorin-3B SEMA3B
Q13219 Pappalysin-1 PAPPA
Q13231 Chitotriosidase-1 CHIT1 Q13253 Noggin NOG
Q13261 Interleukin-15 receptor subunit alpha IL15RA
Q13275 Semaphorin-3F SEMA3F
Q13291 Signaling lymphocytic activation molecule SLAMF1
158 Q13316 Dentin matrix acidic phosphoprotein 1 DMP1 Q13361 Microfibrillar- associated protein 5 MFAP5 Q13410 Butyrophilin subfamily 1 member Al BTN1A1 Q13421 Mesothelin, cleaved form MSLN
Q13429 Insulin-like growth factor I IGF-I
Disintegrin and metalloproteinase domain-containing protein 9 Q13519 Neuropeptide 1 PNOC
Q13751 Laminin subunit beta-3 LAMB3 Q13753 Laminin subunit gamma-2 LAMC2 Q13790 Apolipoprotein F APOF
Ectonucleotide Q13822 pyrophosphatase/phosphodiesterase family ENPP2 member 2 Q14031 Collagen alpha-6(IV) chain COL4A6 Q14050 Collagen alpha-3(IX) chain COL9A3 Q14055 Collagen alpha-2(IX) chain COL9A2 Q14112 Nidogen-2 NID2 Q14114 Low-density lipoprotein receptor-related protein 8 LRP8 Q14118 Dystroglycan DAG1 Q14314 Fibroleukin FGL2 Q14393 Growth arrest-specific protein 6 GAS6 Q14406 Chorionic somatomammotropin hormone-like 1 CSHL1 Q14507 Epididymal secretory protein E3-alpha EDDM3A
Q14508 WAP four-disulfide core domain protein 2 WFDC2 Q14512 Fibroblast growth factor-binding protein 1 FGFBP1 Q14515 SPARC-like protein 1 SPARCL1 Q14520 Hyaluronan-binding protein 2 27 kDa light chain HABP2 Q14563 Semaphorin-3A SEMA3A
Q14623 Indian hedgehog protein IHH
Q14624 Inter-alpha-trypsin inhibitor heavy chain H4 ITIH4 Q14667 UPF0378 protein KIAA0100 KIAA0100 Membrane-bound transcription factor site-1 protease Latent-transforming growth factor beta-binding protein 1 Latent-transforming growth factor beta-binding protein 2 Q14773 Intercellular adhesion molecule 4 ICAM4 Q14993 Collagen alpha-1(XIX) chain COL19A1 Q14CN2 Calcium-activated chloride channel regulator 4' CLCA4 110 kDa form
159 Q15046 Lysine-ARNA ligase KARS
Q15063 Periostin POSTN
Advanced glycosylation end product-specific receptor Q15113 Procollagen C-endopeptidase enhancer 1 PCOLCE
Q15166 Serum paraoxonase/lactonase 3 PON3 Q15195 Plasminogen-like protein A PLGLA
Q15198 Platelet-derived growth factor receptor-like protein PDGFRL
Q15223 Poliovirus receptor-related protein 1 PVRL1 Q15238 Pregnancy-specific beta-l-glycoprotein 5 PSG5 Transmembrane emp24 domain-containing protein Q15375 Ephrin type-A receptor 7 EPHA7 Q15389 Angiopoietin-1 ANGPT1 Q15465 Sonic hedgehog protein SHH
Q15485 Ficolin-2 FCN2 Q15517 Corneodesmosin CDSN
Transforming growth factor-beta-induced protein ig-h3 Q15661 Tryptase alpha/beta-1 TPSAB1 Q15726 Metastin KISS1 Q15782 Chitinase-3-like protein 2 CHI3L2 Q15828 Cystatin-M CST6 Q15846 Clusterin-like protein 1 CLUL1 Q15848 Adiponectin ADIPOQ
Q16206 Protein disulfide-thiol oxidoreductase ENOX2 Q16270 Insulin-like growth factor-binding protein 7 IGFBP7 Q16363 Laminin subunit alpha-4 LAMA4 Q16378 Proline-rich protein 4 PRR4 Q16557 Pregnancy-specific beta-l-glycoprotein 3 PSG3 Q16568 CART(42-89) CARTPT
Q16610 Extracellular matrix protein 1 ECM1 Q16619 Cardiotrophin-1 CTF1 Q16623 Syntaxin-1A STX1A
Q16627 HCC-1(9-74) CCL14 Q16651 Prostasin light chain PRSS8 Q16661 Guanylate cyclase C-activating peptide 2 GUCA2B
Q16663 CCL15(29-92) CCL15 Q16674 Melanoma-derived growth regulatory protein MIA
Q16769 Glutaminyl-peptide cyclotransferase QPCT
160 Q16787 Laminin subunit alpha-3 LAMA3 CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 2 Ql7RR3 Pancreatic lipase-related protein 3 PNLIPRP3 Ql7RW2 Collagen alpha-1(XXIV) chain COL24A1 Ql7RY6 Lymphocyte antigen 6K LY6K
Q1L6U9 Prostate-associated microseminoprotein MSMP
Q1W4C9 Serine protease inhibitor Kazal-type 13 SPINK13 Q1ZYL8 Izumo sperm-egg fusion protein 4 IZUM04 HLA class I histocompatibility antigen, Cw-16 alpha chain Q2I0M5 R-spondin-4 RSPO4 Q2L4Q9 Serine protease 53 PRSS53 Q2MKA7 R-spondin-1 RSPO1 Q2MV58 Tectonic-1 TCTN1 Q2TAL6 Brorin VWC2 Q2UY09 Collagen alpha-1(XXVIII) chain COL28A1 Q2VPA4 Complement component receptor 1-like protein CR1L
Carcinoembryonic antigen-related cell adhesion molecule 16 Q30KP8 Beta-defensin 136 DEFB136 Q30KP9 Beta-defensin 135 DEFB135 Q3 OKQ1 Beta-defensin 133 DEFB133 Q3OKQ2 Beta-defensin 130 DEFB130 Q3OKQ4 Beta-defensin 116 DEFB116 Q3OKQ5 Beta-defensin 115 DEFB115 Q3OKQ6 Beta-defensin 114 DEFB114 Q3OKQ7 Beta-defensin 113 DEFB113 Q3OKQ8 Beta-defensin 112 DEFB112 Q3OKQ9 Beta-defensin 110 DEFB110 Q3OKR1 Beta-defensin 109 DEFB109P1 Q32P28 Prolyl 3-hydroxylase 1 LEPRE1 Glucose-fructose oxidoreductase domain-containing protein 2 Q3SY79 Protein Wnt WNT3A
N-acetylglucosamine-l-phosphotransferase subunits alpha/beta Q495T6 Membrane metallo-endopeptidase-like 1 MMEL1 Q49AHO Cerebral dopamine neurotrophic factor CDNF
Q4G0G5 Secretoglobin family 2B member 2 SCGB2B2 Q4G0M1 Protein FAM132B FAM132B
161 Sushi, von Willebrand factor type A, EGF and pentraxin domain-containing protein 1 Q4QY38 Beta-defensin 134 DEFB134 Q4VAJ4 Protein Wnt WNT1OB
Q4W5P6 Protein TMEM155 TMEM155 Q4ZHG4 Fibronectin type III domain-containing protein 1 FNDC1 Q53H76 Phospholipase Al member A PLA1A
Q53RD9 Fibulin-7 FBLN7 Q53 S33 Bo1A-like protein 3 BOLA3 Q5BLP8 Neuropeptide-like protein C4orf48 C4orf48 Q5DT21 Serine protease inhibitor Kazal-type 9 SPINK9 Q5EBL8 PDZ domain-containing protein 11 PDZD11 Q5FYBO Arylsulfatase J ARSJ
Q5FYB1 Arylsulfatase I ARSI
Q5GAN3 Ribonuclease-like protein 13 RNASE13 Q5GAN4 Ribonuclease-like protein 12 RNASE12 Q5GAN6 Ribonuclease-like protein 10 RNASE10 von Willebrand factor A domain-containing protein 2 Q5H8A3 Neuromedin-S NMS
Q5H8C1 FRAS1-related extracellular matrix protein 1 FREM1 Q5IJ48 Protein crumbs homolog 2 CRB2 Q5J5C9 Beta-defensin 121 DEFB121 Q5J537 NHL repeat-containing protein 3 NHLRC3 Q5JTB6 Placenta-specific protein 9 PLAC9 Q5J1J69 Torsin-2A TOR2A
Q5JXM2 Methyltransferase-like protein 24 METTL24 Q5JZY3 Ephrin type-A receptor 10 EPHA 1 0 Q5K4E3 Polyserase-2 PR5536 Q5SRR4 Lymphocyte antigen 6 complex locus protein G5c LY6G5C
Q5T1H1 Protein eyes shut homolog EYS
Q5T4F7 Secreted frizzled-related protein 5 SFRP5 Q5T4W7 Artemin ARTN
Q5T7M4 Protein FAM132A FAM132A
Q5TEH8 Protein Wnt WNT2B
von Willebrand factor A domain-containing protein 5B1 Q5UCC4 ER membrane protein complex subunit 10 EMC10 Abhydrolase domain-containing protein Q5VTL7 Fibronectin type III domain-containing protein 7 FNDC7
162 Q5VUM1 UPF0369 protein C6orf57 C6orf57 Q5VV43 Dyslexia-associated protein KIAA0319 KIAA0319 Q5VWW1 Complement Clq-like protein 3 ClQL3 Q5VXI9 Lipase member N LIPN
Q5VXJ0 Lipase member K LIPK
Q5VXM1 CUB domain-containing protein 2 CDCP2 Q5VYX0 Renalase RNLS
Q5VYY2 Lipase member M LIPM
Q5W186 Cystatin-9 CST9 Q5W5W9 Regulated endocrine-specific protein 18 RESP18 Q5XG92 Carboxylesterase 4A CES4A
Q63HQ2 Pikachurin EGFLAM
Q641Q3 Meteorin-like protein METRNL
Q66K79 Carboxypeptidase Z CPZ
Q685J3 Mucin-17 MUC17 Q68BL7 Olfactomedin-like protein 2A OLFML2A
Q68BL8 Olfactomedin-like protein 2B OLFML2B
Q68DV7 E3 ubiquitin-protein ligase RNF43 RNF43 Q6B9Z1 Insulin growth factor-like family member 4 IGFL4 Q6BAA4 Fc receptor-like B FCRLB
Q6E0U4 Dermokine DMKN
Q6EMK4 Vasorin VASN
Q6FHJ7 Secreted frizzled-related protein 4 SFRP4 Q6GPI1 Chymotrypsin B2 chain B CTRB2 Q6GTS8 Probable carboxypeptidase PM20D1 PM20D1 Q6H9L7 Isthmin-2 I5M2 Q6IE36 Ovostatin homolog 2 0V052 Q6IE37 Ovostatin homolog 1 OVOS1 Q6IE38 Serine protease inhibitor Kazal-type 14 SPINK14 Leukocyte-associated immunoglobulin-like receptor 2 Q6JVE5 Epididymal-specific lipocalin-12 LCN12 Q6JVE6 Epididymal-specific lipocalin-10 LCN10 Q6JVE9 Epididymal-specific lipocalin-8 LCN8 Q6KF10 Growth/differentiation factor 6 GDF6 Q6MZW2 Follistatin-related protein 4 FSTL4 Q6NSX1 Coiled-coil domain-containing protein 70 CCDC70 Q6NT32 Carboxylesterase 5A CES5A
Q6NT52 Choriogonadotropin subunit beta variant 2 CGB2
163 Q6NUI6 Chondroadherin-like protein CHADL
Q6NUJ1 Saposin A-like PSAPL1 Q6P093 Arylacetamide deacetylase-like 2 AADACL2 Q6P4A8 Phospholipase B-like 1 PLBD1 Q6P5S2 UPF0762 protein C6orf58 C6orf58 Q6P988 Protein notum homolog NOTUM
von Willebrand factor A domain-containing protein 1 Q6PDA7 Sperm-associated antigen 11A SPAG1 1A
Q6PEWO Inactive serine protease 54 PRSS54 Q6PEZ8 Podocan-like protein 1 PODNL1 Dehydrogenase/reductase SDR family member 4-like 2 Q6Q788 Apolipoprotein A-V AP0A5 Q6SPF0 Atherin SAMD1 Q6UDR6 Kunitz-type protease inhibitor 4 SPINT4 Q6URK8 Testis, prostate and placenta-expressed protein TEPP
Q6UW01 Cerebellin-3 CBLN3 Q6UW10 Surfactant-associated protein 2 SFTA2 Q6UW15 Regenerating islet-derived protein 3-gamma REG3G
Q6UW32 Insulin growth factor-like family member 1 IGFL1 Q6UW78 UPF0723 protein Cl lorf83 Cl lorf83 Q6UW88 Epigen EPGN
Q6UWE3 Colipase-like protein 2 CLPSL2 Q6UWF7 NXPE family member 4 NXPE4 Q6UWF9 Protein FAM180A FAM180A
Q6UWM5 GLIPR1-like protein 1 GLIPR1L1 Q6UWN8 Serine protease inhibitor Kazal-type 6 SPINK6 Q6UWP2 Dehydrogenase/reductase SDR family member 11 DHRS11 Q6UWP8 Suprabasin SBSN
Q6UWQ5 Lysozyme-like protein 1 LYZL1 Q6UWQ7 Insulin growth factor-like family member 2 IGFL2 Ectonucleotide Q6UWR7 pyrophosphatase/phosphodiesterase family ENPP6 member 6 soluble form Q6UWT2 Adropin ENHO
Q6UWU2 Beta-galactosidase-l-like protein GLB1L
Q6UWW0 Lipocalin-15 LCN15 Q6UWX4 HHIP-like protein 2 HHIPL2 Q6UWY0 Arylsulfatase K ARSK
Q6UWY2 Serine protease 57 PRSS57
164 Q6UWY5 Olfactomedin-like protein 1 OLFML1 Q6UX06 Olfactomedin-4 0LFM4 Q6UX07 Dehydrogenase/reductase SDR family member 13 DHRS13 Q6UX39 Amelotin AMTN
Q6UX46 Protein FAM150B FAM150B
Q6UX73 UPF0764 protein C16orf89 Cl6orf89 Q6UXBO Protein FAM131A FAM131A
Q6UXB1 Insulin growth factor-like family member 3 IGFL3 Q6UXB2 VEGF co-regulated chemokine 1 CXCL17 Q6UXF7 C-type lectin domain family 18 member B CLEC18B
Q6UXHO Hepatocellular carcinoma-associated protein TD26 Cl9orf80 Q6UXH1 Cysteine-rich with EGF-like domain protein 2 CRELD2 Collagen and calcium-binding EGF domain-containing protein 1 Q6UXH9 Inactive serine protease PAMR1 PAMR1 Q6UXI7 Vitrin VIT
Q6UXI9 Nephronectin NPNT
Q6UXN2 Trem-like transcript 4 protein TREML4 Q6UXSO C-type lectin domain family 19 member A CLEC19A
Q6UXT8 Protein FAM150A FAM150A
Q6UXT9 Abhydrolase domain-containing protein 15 ABHD15 Q6UXV4 Apolipoprotein 0-like APOOL
Q6UXX5 Inter-alpha-trypsin inhibitor heavy chain H6 ITIH6 Q6UXX9 R-spondin-2 RSPO2 Q6UY14 ADAMTS-like protein 4 ADAMTSL4 Q6UY27 Prostate and testis expressed protein 2 PATE2 Q6W4X9 Mucin-6 MUC6 Q6WN34 Chordin-like protein 2 CHRDL2 Q6WRIO Immunoglobulin superfamily member 10 IGSF10 Q6X4U4 Sclerostin domain-containing protein 1 SOSTDC1 Q6X784 Zona pellucida-binding protein 2 ZPBP2 Q6XE38 Secretoglobin family 1D member 4 SCGB1D4 Q6XPR3 Repetin RPTN
Q6XZBO Lipase member I LIPI
Q6ZMM2 ADAMTS-like protein 5 ADAMTSL5 Thrombospondin type-1 domain-containing protein 4 Q6ZNFO Iron/zinc purple acid phosphatase-like protein PAPL
Q6ZRIO Otogelin OTOG
Q6ZRP7 Sulfhydryl oxidase 2 QS0X2
165 Q6ZWJ8 Kielin/chordin-like protein KCP
Q75N90 Fibrillin-3 FBN3 Q76510 Urotensin-2B UTS2D
Q76B58 Protein FAM5C FAM5C
A disintegrin and metalloproteinase with thrombospondin motifs 13 Q76M96 Coiled-coil domain-containing protein 80 CCDC80 Q7L1 S5 Carbohydrate sulfotransferase 9 CHST9 Q7L513 Fc receptor-like A FCRLA
Q7L8A9 Vasohibin-1 VASH1 Q7RTM1 Otopetrin-1 OTOP1 Q7RTW8 Otoancorin OTOA
Q7RTY5 Serine protease 48 PRSS48 Q7RTY7 Ovochymase-1 OVCH1 Q7RTZ1 Ovochymase-2 OVCH2 Q7Z304 MAM domain-containing protein 2 MAMDC2 Q7Z3S9 Notch homolog 2 N-terminal-like protein NOTCH2NL
Q7Z4H4 Intermedin-short ADM2 Q7Z4P5 Growth/differentiation factor 7 GDF7 Q7Z4R8 UPF0669 protein C6orf120 C6orf120 Q7Z4W2 Lysozyme-like protein 2 LYZL2 Q7Z5A4 Serine protease 42 PRSS42 Q7Z5A7 Protein FAM19A5 FAM19A5 Q7Z5A8 Protein FAM19A3 FAM19A3 Q7Z5A9 Protein FAM19A1 FAM19A1 Hydroxysteroid 11-beta-dehydrogenase 1-like protein Q7Z5L0 Vitelline membrane outer layer protein 1 homolog VM01 Q7Z5L3 Complement Clq-like protein 2 C1QL2 Q7Z5L7 Podocan PODN
Q7Z5P4 17-beta-hydroxysteroid dehydrogenase 13 HSD17B13 Q7Z5P9 Mucin-19 MUC19 Q7Z5Y6 Bone morphogenetic protein 8A BMP8A
Q7Z7B7 Beta-defensin 132 DEFB132 Q7Z7B8 Beta-defensin 128 DEFB128 Q7Z7C8 Transcription initiation factor TFIID subunit 8 TAF8 Transmembrane emp24 domain-containing protein Q86SG7 Lysozyme g-like protein 2 LYG2 Q86SI9 Protein CEI C5or138
166 Q86TE4 Leucine zipper protein 2 LUZP2 Q86TH1 ADAMTS-like protein 2 ADAMTSL2 Q86U17 Serpin All SERPINAll Q86U1J9 Endokinin-A TAC4 Q86UW8 Hyaluronan and proteoglycan link protein 4 HAPLN4 Q86UX2 Inter-alpha-trypsin inhibitor heavy chain H5 ITIH5 Q86V24 Adiponectin receptor protein 2 ADIPOR2 Q86VB7 Soluble CD163 CD163 Q86VR8 Four-jointed box protein 1 FJX1 Q86WD7 Serpin A9 SERPINA9 Q86WN2 Interferon epsilon IFNE
Q86W53 Placenta-specific 1-like protein PLAC1L
Q86X52 Chondroitin sulfate synthase 1 CHSY1 Q86XP6 Gastrokine-2 GKN2 Q86X55 Angiopoietin-related protein 5 ANGPTL5 Q86Y27 B melanoma antigen 5 BAGE5 Q86Y28 B melanoma antigen 4 BAGE4 Q86Y29 B melanoma antigen 3 BAGE3 Q86Y30 B melanoma antigen 2 BAGE2 Q86Y38 Xylosyltransferase 1 XYLT1 Q86Y78 Ly6/PLAUR domain-containing protein 6 LYPD6 Q86YD3 Transmembrane protein 25 TMEM25 Q86YJ6 Threonine synthase-like 2 THNSL2 Q86YW7 Glycoprotein hormone beta-5 GPHB5 Q86Z23 Complement Clq-like protein 4 ClQL4 Q8IU57 Interleukin-28 receptor subunit alpha IL28RA
Q8IUAO WAP four-disulfide core domain protein 8 WFDC8 Q8IUB2 WAP four-disulfide core domain protein 3 WFDC3 Q8IUB3 Protein WFDC1OB WFDC1OB
Q8IUB5 WAP four-disulfide core domain protein 13 WFDC13 Q8IUH2 Protein CREG2 CREG2 Q8IUK5 Plexin domain-containing protein 1 PLXDC1 Q8IUL8 Cartilage intermediate layer protein 2 C2 CILP2 Q8IUX7 Adipocyte enhancer-binding protein 1 AEBP1 Q8IUX8 Epidermal growth factor-like protein 6 EGFL6 Q8IVL8 Carboxypeptidase 0 CPO
Somatomedin-B and thrombospondin type-1 domain-containing protein Q8IVW8 Protein spinster homolog 2 SPNS2
167 Q8IW75 Serpin Al2 SERPINA12 Q8IW92 Beta-galactosidase-l-like protein 2 GLB1L2 Q8IWL1 Pulmonary surfactant-associated protein A2 SFTPA2 Q8IWL2 Pulmonary surfactant-associated protein Al SFTPA1 Q8IWV2 Contactin-4 CNTN4 Signal peptide, CUB and EGF-like domain-containing protein 1 Signal peptide, CUB and EGF-like domain-containing protein 3 Sperm acrosome membrane-associated protein 3,Q8IXA5 SPACA3 membrane form Q8IXB1 DnaJ homolog subfamily C member 10 DNAJC10 Extracellular serine/threonine protein kinase Fam20C
Q8IYD9 Lung adenoma susceptibility protein 2 LAS2 Q8IYP2 Serine protease 58 PR5558 Osteoclast-associated immunoglobulin-like receptor Q8IZC6 Collagen alpha-1(XXVII) chain COL27A1 C3 and PZP-like alpha-2-macroglobulin domain-containing protein 8 Q8IZN7 Beta-defensin 107 DEFB107B
Q8NOV4 Leucine-rich repeat LGI family member 2 LGI2 Q8N104 Beta-defensin 106 DEFB106B
Q8N119 Matrix metalloproteinase-21 MMP21 Q8N129 Protein canopy homolog 4 CNPY4 Q8N135 Leucine-rich repeat LGI family member 4 LGI4 Q8N145 Leucine-rich repeat LGI family member 3 LGI3 Q8N158 Glypican-2 GPC2 Q8N1E2 Lysozyme g-like protein 1 LYG1 von Willebrand factor D and EGF domain-containing protein Q8N2E6 Prosalusin TOR2A
Latent-transforming growth factor beta-binding protein 4 Angiogenic factor with G patch and FHA domains Q8N307 Mucin-20 MUC20 Q8N323 NXPE family member 1 NXPE1 Q8N387 Mucin-15 MUC15 Q8N3Z0 Inactive serine protease 35 PR5535 Q8N436 Inactive carboxypeptidase-like protein X2 CPXM2 Q8N474 Secreted frizzled-related protein 1 SF RP1 Q8N475 Follistatin-related protein 5 FSTL5
168 Q8N4F0 BPI fold-containing family B member 2 BPIFB2 Q8N4TO Carboxypeptidase A6 CPA6 Q8N5W8 Protein FAM24B FAM24B
Q8N687 Beta-defensin 125 DEFB125 Q8N688 Beta-defensin 123 DEFB123 Q8N690 Beta-defensin 119 DEFB119 Q8N6C5 Immunoglobulin superfamily member 1 IGSF1 Leukocyte immunoglobulin-like receptor subfamily A member 3 Q8N6G6 ADAMTS-like protein 1 ADAMTSL1 Q8N6Y2 Leucine-rich repeat-containing protein 17 LRRC17 Q8N729 Neuropeptide W-23 NPW
Q8N8U9 BMP-binding endothelial regulator protein BMPER
Q8N907 DAN domain family member 5 DAND5 Glycosyltransferase-like domain-containing protein 2 Q8NAU1 Fibronectin type III domain-containing protein 5 FNDC5 Q8NB37 Parkinson disease 7 domain-containing protein 1 PDDC1 Q8NBI3 Draxin DRAXIN
Q8NBM8 Prenylcysteine oxidase-like PCY0X1L
Q8NBP7 Proprotein convertase subtilisin/kexin type 9 PCSK9 Q8NBQ5 Estradiol 17-beta-dehydrogenase 11 HSD17B11 Q8NBV8 Synaptotagmin-8 SYT8 Q8NCC3 Group XV phospholipase A2 PLA2G15 Q8NCF0 C-type lectin domain family 18 member C CLEC18C
Q8NCW5 NAD(P)H-hydrate epimerase AP0A1BP
Q8NDA2 Hemicentin-2 HMCN2 Q8NDX9 Lymphocyte antigen 6 complex locus protein G5b LY6G5B
Q8NDZ4 Deleted in autism protein 1 C3orf58 Q8NEB7 Acrosin-binding protein ACRBP
Q8NES8 Beta-defensin 124 DEFB124 Q8NET1 Beta-defensin 108B DEFB108B
Q8NEX5 Protein WFDC9 WFDC9 Q8NEX6 Protein WFDC11 WFDC11 Q8NF86 Serine protease 33 PRSS33 Q8NFM7 Interleukin-17 receptor D IL17RD
Q8NFQ5 BPI fold-containing family B member 6 BPIFB6 Q8NFQ6 BPI fold-containing family C protein BPIFC
Q8NFU4 Follicular dendritic cell secreted peptide FDCSP
Q8NFW1 Collagen alpha-1(XXII) chain COL22A1
169 Q8NG35 Beta-defensin 105 DEFB105B
Q8NG41 Neuropeptide B-23 NPB
Q8NHW6 Otospiralin OTOS
Q8NI99 Angiopoietin-related protein 6 ANGPTL6 Q8TAA1 Probable ribonuclease 11 RNASEll V-set and transmembrane domain-containing protein 2A
Q8TAL6 Fin bud initiation factor homolog FIBIN
Q8TAT2 Fibroblast growth factor-binding protein 3 FGFBP3 Q8TAX7 Mucin-7 MUC7 Q8TB22 Spermatogenesis-associated protein 20 SPATA20 Q8TB73 Protein NDNF NDNF
Q8TB96 T-cell immunomodulatory protein ITFG1 Q8TC92 Protein disulfide-thiol oxidoreductase ENOX1 Q8TCV5 WAP four-disulfide core domain protein 5 WFDC5 Q8TD06 Anterior gradient protein 3 homolog AGR3 Q8TD33 Secretoglobin family 1C member 1 SCGB1C1 Q8TD46 Cell surface glycoprotein CD200 receptor 1 CD200R1 Q8TDE3 Ribonuclease 8 RNASE8 Q8TDF5 Neuropilin and tolloid-like protein 1 NET01 Q8TDL5 BPI fold-containing family B member 1 BPIFB1 A disintegrin and metalloproteinase with thrombospondin motifs 17 A disintegrin and metalloproteinase with thrombospondin motifs 16 A disintegrin and metalloproteinase with thrombospondin motifs 15 A disintegrin and metalloproteinase with thrombospondin motifs 19 A disintegrin and metalloproteinase with thrombospondin motifs 18 Q8TE99 Acid phosphatase-like protein 2 ACPL2 Sushi, nidogen and EGF-like domain-containing protein 1 WAP, kazal, immunoglobulin, kunitz and NTR

domain-containing protein 2 Q8WTQ1 Beta-defensin 104 DEFB104B
Q8WTR8 Netrin-5 NTN5 Scavenger receptor cysteine-rich domain-containing group B protein Q8WU66 Protein T SPEAR T SPEAR
Q8WUA8 Tsukushin TSKU
Q8WUF8 Protein FAM172A FAM172A
Q8WUJ1 Neuferricin CYB5D2
170 Q8WUY1 UPF0670 protein THEM6 THEM6 Q8WVN6 Secreted and transmembrane protein 1 SECTM1 Q8WVQ1 Soluble calcium-activated nucleotidase 1 CANT1 Q8WWAO Intelectin-1 ITLN1 Q8WWG1 Neuregulin-4 NRG4 Q8WWQ2 Inactive heparanase-2 HPSE2 Q8WWU7 Intelectin-2 ITLN2 Q8WWY7 WAP four-disulfide core domain protein 12 WFDC12 Q8WWY8 Lipase member H LIPH
Q8WWZ8 Oncoprotein-induced transcript 3 protein 0IT3 Q8WX39 Epididymal-specific lipocalin-9 LCN9 Q8WXA2 Prostate and testis expressed protein 1 PATE1 Q8WXD2 Secretogranin-3 SCG3 Q8WXF3 Relaxin-3 A chain RLN3 Q8WXI7 Mucin-16 MUC16 Q8WXQ8 Carboxypeptidase A5 CPAS
A disintegrin and metalloproteinase with thrombospondin motifs 14 Q92484 Acid sphingomyelinase-like phosphodiesterase 3a SMPDL3A
Q92485 Acid sphingomyelinase-like phosphodiesterase 3b SMPDL3B
Q92496 Complement factor H-related protein 4 CFHR4 Q92520 Protein FAM3C FAM3C
Q92563 Testican-2 SPOCK2 Q92583 C-C motif chemokine 17 CCL17 Q92626 Peroxidasin homolog PXDN
Q92743 Serine protease HTRA1 HTRA1 Q92752 Tenascin-R TNR
Q92765 Secreted frizzled-related protein 3 FRZB
Q92819 Hyaluronan synthase 2 HAS2 Q92820 Gamma-glutamyl hydrolase GGH
Q92824 Proprotein convertase subtilisinikexin type 5 PCSK5 Q92832 Protein kinase C-binding protein NELL1 NELL1 Q92838 Ectodysplasin-A, membrane form EDA
Q92874 Deoxyribonuclease-l-like 2 DNASE1L2 Q92876 Kallikrein-6 KLK6 Q92913 Fibroblast growth factor 13 FGF13 Q92954 Proteoglycan 4 C-terminal part PRG4 Tumor necrosis factor receptor superfamily member 25 Q93091 Ribonuclease K6 RNASE6
171 Q93097 Protein Wnt-2b WNT2B
Q93098 Protein Wnt-8b WNT8B
Major histocompatibility complex class I-related gene protein Q969D9 Thymic stromal lymphopoietin TSLP
Q969E1 Liver-expressed antimicrobial peptide 2 LEAP2 Q969H8 UPF0556 protein Cl9orf10 Cl9orf10 Q969Y0 NXPE family member 3 NXPE3 Q96A54 Adiponectin receptor protein 1 ADIPOR1 Q96A83 Collagen alpha-1(XXVI) chain EMID2 Q96A84 EMI domain-containing protein 1 EMID1 Q96A98 Tuberoinfundibular peptide of 39 residues PTH2 Q96A99 Pentraxin-4 PTX4 Q96BH3 Epididymal sperm-binding protein 1 ELSPBP1 Q96BQ1 Protein FAM3D FAM3D
Q96CG8 Collagen triple helix repeat-containing protein 1 CTHRC1 Q96DA0 Zymogen granule protein 16 homolog B ZG16B
von Willebrand factor C and EGF domain-containing protein Q96DR5 BPI fold-containing family A member 2 BPIFA2 Q96DR8 Mucin-like protein 1 MUCL1 RING finger and SPRY domain-containing protein Q96EE4 Coiled-coil domain-containing protein 126 CCDC126 Abhydrolase domain-containing protein Q96GW7 Brevican core protein BCAN
Q96HF1 Secreted frizzled-related protein 2 SFRP2 Kazal-type serine protease inhibitor domain-containing protein 1 Q96ID5 Immunoglobulin superfamily member 21 IGSF21 Leucine-rich repeat and calponin homology domain-containing protein 3 Q96IY4 Carboxypeptidase B2 CPB2 Q96JB6 Lysyl oxidase homolog 4 LOXL4 Q96JK4 HHIP-like protein 1 HHIPL1 Q96KN2 Beta-Ala-His dipeptidase CNDP1 Q96KW9 Protein SPACA7 SPACA7 Q96KX0 Lysozyme-like protein 4 LYZL4 Q96L15 Ecto-ADP-ribosyltransferase 5 ARTS
Q96LB8 Peptidoglycan recognition protein 4 PGLYRP4 Q96LB9 Peptidoglycan recognition protein 3 PGLYRP3
172 Q96LC7 Sialic acid-binding Ig-like lectin 10 SIGLEC10 Q96LR4 Protein FAM19A4 FAM19A4 Q96MK3 Protein FAM20A FAM20A
Q96MS3 Glycosyltransferase 1 domain-containing protein 1 GLT1D1 Q96NY8 Processed poliovirus receptor-related protein 4 PVRL4 WAP, kazal, immunoglobulin, kunitz and NTR

domain-containing protein 1 Q96NZ9 Proline-rich acidic protein 1 PRAP1 Q96P44 Collagen alpha-1(XXI) chain COL21A1 Q96PB7 Noelin-3 OLFM3 Q96PC5 Melanoma inhibitory activity protein 2 MIA2 Q96PD5 N-acetylmuramoyl-L-alanine amidase PGLYRP2 Q96PH6 Beta-dcfensin 118 DEFB118 Q96PL1 Secretoglobin family 3A member 2 SCGB3A2 Q96PL2 Beta-tectorin TECTB
Q96QH8 Sperm acrosome-associated protein 5 SPACA5 Q96QR1 Secretoglobin family 3A member 1 SCGB3A1 Q96QU1 Protocadherin-15 PCDH15 Q96QV1 Hedgehog-interacting protein HHIP
Q96RW7 Hemicentin-1 HMCN1 Q96S42 Nodal homolog NODAL
Q96S86 Hyaluronan and proteoglycan link protein 3 HAPLN3 Q96SL4 Glutathione peroxidase 7 GPX7 Q965M3 Probable carboxypeptidase X1 CPXM1 Q96T91 Glycoprotein hormone alpha-2 GPHA2 Q99062 Granulocyte colony-stimulating factor receptor CSF3R
Q99102 Mucin-4 alpha chain MUC4 Q99217 Amelogenin, X isoform AMELX
Q99218 Amelogenin, Y isoform AMELY
Q99435 Protein kinase C-binding protein NELL2 NELL2 Q99470 Stromal cell-derived factor 2 SDF2 Q99542 Matrix metalloproteinase-19 MMP19 Q99574 Neuroserpin SERPINI1 Q99584 Protein S100-A13 S100A13 Q99616 C-C motif chemokine 13 CCL13 Q99645 Epiphycan EPYC
Cell growth regulator with EF hand domain protein 1 Q99715 Collagen alpha-1(XII) chain COL12A1 Q99727 Metalloproteinase inhibitor 4 TIMP4
173 Q99731 C-C motif chemokine 19 CCL19 Q99748 Neurturin NRTN
Q99935 Proline-rich protein 1 PROL1 Q99942 E3 ubiquitin-protein ligase RNF5 RNF5 Q99944 Epidermal growth factor-like protein 8 EGFL8 Q99954 Submaxillary gland androgen-regulated protein 3A SMR3A
Q99969 Retinoic acid receptor responder protein 2 RARRES2 Q99972 Myocilin MYOC
Q99983 Osteomodulin OMD
Q99985 Semaphorin-3C SEMA3C
Q99988 Growth/differentiation factor 15 GDF15 Q9BPW4 Apolipoprotein L4 APOL4 Q9BQ08 Resistin-like beta RETNLB
Q9BQ16 Testican-3 SPOCK3 Q9BQ51 Programmed cell death 1 ligand 2 PDCD1LG2 Q9BQB4 Sclerostin SOST
Q9BQI4 Coiled-coil domain-containing protein 3 CCDC3 Q9BQP9 BPI fold-containing family A member 3 BPIFA3 Q9BQR3 Serine protease 27 PR5527 Q9BQY6 WAP four-disulfide core domain protein 6 WFDC6 Q9BRR6 ADP-dependent glucokinase ADPGK
Q9B586 Zona pellucida-binding protein 1 ZPBP
Q9BSGO Protease-associated domain-containing protein 1 PRADC1 Q9BSG5 Retbindin RTBDN
Probable alpha-ketoglutarate-dependent dioxygenase ABH7 Q9BT56 Spexin C12or139 Q9BT67 NEDD4 family-interacting protein 1 NDFIP1 Q9BTY2 Plasma alpha-L-fucosidase FUCA2 Q9BU40 Chordin-like protein 1 CHRDL1 Q9BUD6 Spondin-2 SPON2 Q9BUN1 Protein MENT MENT
Q9BUR5 Apolipoprotein 0 APOO
ER degradation-enhancing alpha-mannosidase-like Q9BWP8 Collectin-11 COLEC11 Q9BWS9 Chitinase domain-containing protein 1 CHID1 Q9BX67 Junctional adhesion molecule C JAM3 Group XIIB secretory phospholipase A2-like protein
174 Complement Clq tumor necrosis factor-related protein 6 Complement Clq tumor necrosis factor-related protein 5 Complement Clq tumor necrosis factor-related protein 1 Complement Clq tumor necrosis factor-related protein 7 Complement Clq tumor necrosis factor-related protein 4 Complement Clq tumor necrosis factor-related protein 3 Complement Clq tumor necrosis factor-related protein 2 Q9BXN1 Asporin ASPN
Q9BXP8 Pappalysin-2 PAPPA2 Q9BXR6 Complement factor H-related protein 5 CFHR5 Q9BXSO Collagen alpha-1(XXV) chain COL25A1 Q9BXY4 R-spondin-3 RSPO3 EGF-like module-containing mucin-like hormone receptor-like 3 subunit beta Signal peptidase complex catalytic subunit Q9BY76 Angiopoietin-related protein 4 ANGPTL4 Q9BYF1 Processed angiotensin-converting enzyme 2 ACE2 Q9BYJO Fibroblast growth factor-binding protein 2 FGFBP2 Q9BYW3 Beta-defensin 126 DEFB126 Interferon-induced helicase C domain-containing protein 1 Q9BYZ8 Regenerating islet-derived protein 4 REG4 Q9BZ76 Contactin-associated protein-like 3 CNTNAP3 Q9BZG9 Ly-6/neurotoxin-like protein 1 LYNX1 Q9BZJ3 Tryptase delta TPSD1 Q9BZM1 Group XIIA secretory phospholipase A2 PLA2G12A
Q9BZM2 Group IIF secretory phospholipase A2 PLA2G2F
Q9BZM5 NKG2D ligand 2 ULBP2 Q9BZP6 Acidic mammalian chitinase CHIA
Q9BZZ2 Sialoadhesin SIGLEC1 Q9C0B6 Protein FAM5B FAM5B
Q9GZM7 Tubulointerstitial nephritis antigen-like TINAGL1 Q9GZN4 Brain-specific serine protease 4 PR5522 Platelet-derived growth factor D, receptor-binding form Q9GZT5 Protein Wnt-1 0 a WNT1 OA
175 Q9GZU5 Nyctalopin NYX
Q9GZV7 Hyaluronan and proteoglycan link protein 2 HAPLN2 Q9GZV9 Fibroblast growth factor 23 FGF23 Q9GZX9 Twisted gastrulation protein homolog 1 TWSG1 Q9GZZ7 GDNF family receptor alpha-4 GFRA4 Q9GZZ8 Extracellular glycoprotein lacritin LACRT
Cysteine-rich secretory protein LCCL domain-containing 2 Q9H106 Signal-regulatory protein delta SIRPD
Q9H114 Cystatin-like 1 CSTL1 Q9H173 Nucleotide exchange factor SIL1 SIL1 Q9H1E1 Ribonuclease 7 RNASE7 Q9H1F0 WAP four-disulfide core domain protein 10A WFDC10A
Q9H1J5 Protein Wnt-8a WNT8A
Q9H1J7 Protein Wnt-5b WNT5B
Q9H1M3 Beta-defensin 129 DEFB129 Q9H1M4 Beta-defensin 127 DEFB127 Q9H1Z8 Augurin C2orf40 Q9H239 Matrix metalloproteinase-28 MMP28 Q9H2A7 C-X-C motif chemokine 16 CXCL16 Q9H2A9 Carbohydrate sulfotransferase 8 CHST8 Q9H2R5 Kallikrein-15 KLK15 Q9H2X0 Chordin CHRD
Q9H2X3 C-type lectin domain family 4 member M CLEC4M
Q9H306 Matrix metalloproteinase-27 MMP27 A disintegrin and metalloproteinase with thrombospondin motifs 10 Cysteine-rich secretory protein LCCL domain-containing 1 Q9H3E2 Sorting nexin-25 5NX25 Q9H3R2 Mucin-13 MUC13 Q9H3U7 SPARC-related modular calcium-binding protein 2 SMOC2 Q9H3Y0 Peptidase inhibitor R3HDML R3HDML
Q9H4A4 Aminopeptidase B RNPEP
Q9H4F8 SPARC-related modular calcium-binding protein 1 SMOC1 Q9H4G1 Cystatin-9-like CST9L
Q9H5V8 CUB domain-containing protein 1 CDCP1 Q9H6B9 Epoxide hydrolase 3 EPHX3 Q9H6E4 Coiled-coil domain-containing protein 134 CCDC134 Q9H741 UPF0454 protein C12orf49 C12orf49
176 Q9H772 Gremlin-2 GREM2 Q9H7Y0 Deleted in autism-related protein 1 CXorf36 Q9H8L6 Multimerin-2 MMRN2 Q9H9S5 Fukutin-related protein FKRP
Q9HAT2 Sialate 0-acetylesterase SIAE
Q9HB40 Retinoid-inducible serine carboxypeptidase SCPEP1 Q9HB63 Netrin-4 NTN4 Q9HBJ0 Placenta-specific protein 1 PLAC1 Q9HC23 Prokineticin-2 PROK2 Q9HC57 WAP four-disulfide core domain protein 1 WFDC1 Q9HC73 Cytokine receptor-like factor 2 CRLF2 Q9HC84 Mucin-5B MUC5B
Q9HCB6 Spondin-1 SPON1 Q9HCQ7 Neuropeptide NPSF NPVF
Q9HCTO Fibroblast growth factor 22 FGF22 Q9HD89 Resistin RETN
Q9NNX1 Tuftelin TUFT1 Q9NNX6 CD209 antigen CD209 Q9NP55 BPI fold-containing family A member 1 BPIFA1 Q9NP70 Ameloblastin AMBN
Q9NP95 Fibroblast growth factor 20 FGF20 Q9NP99 Triggering receptor expressed on myeloid cells 1 TREM1 Q9NPA2 Matrix metalloproteinase-25 MMP25 Q9NPE2 Neugrin NGRN
Q9NPHO Lysophosphatidic acid phosphatase type 6 ACP6 Q9NPH6 Odorant-binding protein 2b OBP2B
Q9NQ30 Endothelial cell-specific molecule 1 ESM1 Signal peptide, CUB and EGF-like domain-containing protein 2 Q9NQ38 Serine protease inhibitor Kazal-type 5 SPINK5 Q9NQ76 Matrix extracellular phosphoglycoprotein MEPE
Q9NQ79 Cartilage acidic protein 1 CRTAC1 Scavenger receptor cysteine-rich type 1 protein Q9NR23 Growth/differentiation factor 3 GDF3 Q9NR71 Neutral ceramidase ASAH2 Q9NR99 Matrix-remodeling-associated protein 5 MXRA5 Q9NRA1 Platelet-derived growth factor C PDGFC
Q9NRC9 Otoraplin OTOR
Q9NRE1 Matrix metalloproteinase-26 MMP26
177 Q9NRJ3 C-C motif chemokine 28 CCL28 Q9NRM1 Enamelin ENAM
Q9NRN5 Olfactomedin-like protein 3 OLFML3 Q9NRR1 Cytokine-like protein 1 CYTL1 Latent-transforming growth factor beta-binding protein 3 Thrombospondin type-1 domain-containing protein 1 Q9NS71 Gastrokine-1 GKN1 Q9NS98 Semaphorin-3G SEMA3G
Q9NSA1 Fibroblast growth factor 21 FGF21 Q9NTU7 Cerebellin-4 CBLN4 Q9NVRO Kelch-like protein 11 KLHL11 Q9NWH7 Spermatogenesis-associated protein 6 SPATA6 Glucose-fructose oxidoreductase domain-containing protein 1 Q9NY56 Odorant-binding protein 2a OBP2A
Q9NY84 Vascular non-inflammatory molecule 3 VNN3 Q9NZ20 Group 3 secretory phospholipase A2 PLA2G3 Q9NZC2 Triggering receptor expressed on myeloid cells 2 TREM2 Q9NZK5 Adenosine deaminase CECR1 CECR1 Q9NZK7 Group TIE secretory phospholipase A2 PLA2G2E
Q9NZP8 Complement C lr subcomponent-like protein C 1 RL
Q9NZV1 Cysteine-rich motor neuron 1 protein CRIM1 Q9NZW4 Dentin sialoprotein DSPP
Q9P0G3 Kallikrein-14 KLK14 Q9POWO Interferon kappa IFNK
Q9P218 Collagen alpha-1(XX) chain COL20A1 Q9P2C4 Transmembrane protein 181 TMEM181 Q9P2K2 Thioredoxin domain-containing protein 16 TXNDC16 A disintegrin and metalloproteinase with thrombospondin motifs 9 Q9UBC7 Galanin-like peptide GALP
Q9UBD3 Cytokine SCM-1 beta XCL2 Q9UBD9 Cardiotrophin-like cytokine factor 1 CLCF 1 Q9UBM4 Opticin OPTC
Q9UBP4 Dickkopf-related protein 3 DKK3 Q9UBQ6 Exostosin-like 2 EXTL2 Q9UBR5 Chemokine-like factor CKLF
Q9UBS5 Gamma-aminobutyric acid type B receptor subunit GABBR1
178 Q9UBT3 Dickkopf-related protein 4 short form DKK4 Q9UBU2 Dickkopf-related protein 2 DKK2 Q9UBU3 Ghrelin-28 GHRL
Q9UBV4 Protein Wnt-16 WNT16 Q9UBX5 Fibulin-5 FBLN5 Q9UBX7 Kallikrein-11 KLK11 Q9UEF7 Klotho KL
Q9UFP1 Protein FAM198A FAM198A
Q9UGM3 Deleted in malignant brain tumors 1 protein DMBT1 Q9UGM5 Fetuin-B FETUB
Q9UGP8 Translocation protein SEC63 homolog SEC63 Q9UHFO Neurokinin-B TAC3 Q9UHF1 Epidermal growth factor-like protein 7 EGFL7 Q9UHG2 ProSAAS PCSK1N
A disintegrin and metalloproteinase with thrombospondin motifs 1 Q9UHL4 Dipeptidyl peptidase 2 DPP7 Q9UI42 Carboxypeptidase A4 CPA4 Q9UIG4 Psoriasis susceptibility 1 candidate gene 2 protein PSORS1C2 Q9UIK5 Tomoregulin-2 TMEFF2 Leucyl-cystinyl aminopeptidase, pregnancy serum form Ectonucleotide Q9UJA9 pyrophosphatase/phosphodiesterase family ENPP5 member 5 Q9UJH8 Meteorin METRN
N-acetylglucosamine-l-phosphotransferase subunit gamma Q9UJW2 Tubulointerstitial nephritis antigen TINAG
Q9UK05 Growth/differentiation factor 2 GDF2 Q9UK55 Protein Z-dependent protease inhibitor SERPINA10 Q9UK85 Dickkopf-like protein 1 DKKL1 Q9UKJ1 Paired immunoglobulin-like type 2 receptor alpha PILRA
A disintegrin and metalloproteinase with thrombospondin motifs 7 A disintegrin and metalloproteinase with thrombospondin motifs 6 Disintegrin and metalloproteinase domain-containing protein 28 Q9UKQ9 Kallikrein-9 KLK9 Q9UKRO Kallikrein-12 KLK12 Q9UKR3 Kallikrein-13 KLK13
179 Q9UKU9 Angiopoietin-related protein 2 ANGPTL2 Q9UKZ9 Procollagen C-endopeptidase enhancer 2 PCOLCE2 Transmembrane protease serine 11E non-catalytic chain Q9ULCO Endomucin EMCN
Q9ULI3 Protein HEG homolog 1 HEG1 Q9ULZ1 Apelin-13 APLN
Q9ULZ9 Matrix metalloproteinase-17 MMP17 Alpha-1,3-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase A soluble form Q9UM22 Mammalian ependymin-related protein 1 EPDR1 Q9UM73 ALK tyrosine kinase receptor ALK
Q9UMD9 97 kDa linear IgA disease antigen COL17A1 Q9UMX5 Neudesin NENF
Q9UN73 Protocadherin alpha-6 PCDHA6 A disintegrin and metalloproteinase with thrombospondin motifs 5 Q9UNI1 Chymotrypsin-like elastase family member 1 CELA1 Q9UNK4 Group IID secretory phospholipase A2 PLA2G2D
A disintegrin and metalloproteinase with thrombospondin motifs 8 Thrombospondin type-1 domain-containing protein 7A
Q9UQ72 Pregnancy-specific beta-l-glycoprotein 11 PSG11 Q9UQ74 Pregnancy-specific beta-l-glycoprotein 8 PSG8 Q9UQC9 Calcium-activated chloride channel regulator 2 CLCA2 Q9UQE7 Structural maintenance of chromosomes protein 3 SMC3 Q9UQP3 Tenascin-N TNN
Q9Y223 UDP-N-acetylglucosamine 2-epimerase GNE
Q9Y240 C-type lectin domain family 11 member A CLEC1 1 A
Q9Y251 Heparanase 8 kDa subunit HP SE
Q9Y258 C-C motif chemokine 26 CCL26 Q9Y264 Angiopoietin-4 ANGPT4 Tumor necrosis factor ligand superfamily member 13b, membrane form Q9Y287 BRI2 intracellular domain ITM2B
Q9Y2E5 Epididymis-specific alpha-mannosidase MAN2B2 von Willebrand factor A domain-containing protein 7 Q9Y337 Kallikrein-5 KLK5 Transmembrane emp24 domain-containing protein Q9Y3E2 Bo1A-like protein 1 BOLA1
180 Q9Y426 C2 domain-containing protein 2 C2CD2 Q9Y4K0 Lysyl oxidase homolog 2 LOXL2 Q9Y4X3 C-C motif chemokine 27 CCL27 Q9Y5C1 Angiopoietin-related protein 3 ANGPTL3 Q9Y5I2 Protocadherin alpha-10 PCDHA10 Q9Y5I3 Protocadherin alpha-1 PCDHAl Q9Y5K2 Kallikrein-4 KLK4 Q9Y5L2 Hypoxia-inducible lipid droplet-associated protein HILPDA
Q9Y5Q5 Atrial natriuretic peptide-converting enzyme CORIN
Q9Y5R2 Matrix metalloproteinase-24 MMP24 Tumor necrosis factor receptor superfamily member 18 Q9Y5W5 Wnt inhibitory factor 1 WIF1 Q9Y5X9 Endothelial lipase LIPG
Q9Y625 Secreted glypican-6 GPC6 Q9Y646 Carboxypeptidase Q CPQ

Q9Y6F9 Protein Wnt-6 WNT6 Q9Y6I9 Testis-expressed sequence 264 protein TEX264 Q9Y6L7 Tolloid-like protein 2 TLL2 Calcium-activated chloride channel regulator family member 3 Q9Y6N6 Laminin subunit gamma-3 LAMC3 Q9Y6R7 IgGF c-binding protein FCGBP
Q9Y6Y9 Lymphocyte antigen 96 LY96 Q9Y6Z7 Collectin-10 COLEC10 [0378] In some embodiments, the compositions and methods of the invention provide for the delivery of one or more mRNAs encoding one or more additional exemplary proteins listed in Table 2; thus, compositions of the invention may comprise an mRNA encoding a protein listed in Table 2 (or a homolog thereof) along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA
encoding a protein chosen from the proteins listed in Table 2 (or a homolog thereof) along with other components set out herein.
Table 2. Additional Exemplary Proteins Uniprot ID Protein Name Gene Name
181 A6NGW2 Putative stereocilin-like protein STRCP1 A6NIE9 Putative serine protease 29 PRSS29P
Putative V-set and immunoglobulin domain-containing-like protein IGHV40R15-8 Putative V-set and immunoglobulin domain-containing-like protein IGHV10R21-1 A6NMY6 Putative annexin A2-like protein ANXA2P2 A8MT79 Putative zinc-alpha-2-glycoprotein-like 1 Putative killer cell immunoglobulin-like receptor like protein KIR3DP1 A8MXU0 Putative beta-defensin 108A DEFB108P1 C9JUS6 Putative adrenomedullin-5-like protein ADM5 Putative signal peptidase complex catalytic subunit P00854 Putative cat eye syndrome critical region protein 9 CECR9 Q13046 Putative pregnancy-specific beta-l-glycoprotein 7 PSG7 Q16609 Putative apolipoprotein(a)-like protein 2 LPAL2 Q2TV78 Putative macrophage-stimulating protein MSTP9 MST1P9 Q5JQD4 Putative peptide YY-3 PYY3 Putative inactive group TIC secretory phospholipase A2 Q5VSP4 Putative lipocalin 1-like protein 1 LCN1P1 Q5W188 Putative cystatin-9-like protein CST9LP1 CST9LP1 Q6UXR4 Putative serpin Al3 SERPINA13P
Q865H4 Putative testis-specific prion protein PRNT
Q86YQ2 Putative latherin LATH
Q8IVG9 Putative humanin peptide MT-RNR2 Q8NHM4 Putative trypsin-6 TRY6 Q8NHW4 C-C motif chemokine 4-like CCL4L2 Putative killer cell immunoglobulin-like receptor-like protein KIR3DX1 Q9NRI6 Putative peptide YY-2 PYY2 Q9UF72 Putative TP73 antisense gene protein 1 TP73-AS1 Q9UKY3 Putative inactive carboxylesterase 4 CES1P1 [0379] The Uniprot IDs set forth in Table 1 and Table 2 refer to the human versions the listed proteins and the sequences of each are available from the Uniprot database. Sequences of the listed proteins are also generally available for various animals, including various mammals and animals of veterinary or industrial interest. Accordingly, in some embodiments, compositions and methods of the invention provide for the delivery of one or more mRNAs
182 encoding one or more proteins chosen from mammalian homologs or homologs from an animal of veterinary or industrial interest of the secreted proteins listed in Table 1 or Table 2; thus, compositions of the invention may comprise an mRNA encoding a protein chosen from mammalian homologs or homologs from an animal of veterinary or industrial interest of a protein listed in Table 1 or Table 2 along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA
encoding a protein chosen from mammalian homologs or homologs from an animal of veterinary or industrial interest of a protein listed in Table 1 or Table 2 along with other components set out herein. In some embodiments, mammalian homologs are chosen from mouse, rat, hamster, gerbil, horse, pig, cow, llama, alpaca, mink, dog, cat, ferret, sheep, goat, or camel homologs. In some embodiments, the animal of veterinary or industrial interest is chosen from the mammals listed above and/or chicken, duck, turkey, salmon, catfish, or tilapia.
[0380] In embodiments, the compositions and methods of the invention provide for the delivery of mRNA encoding a lysosomal protein chosen from Table 3. In some embodiments, the compositions and methods of the invention provide for the delivery of one or more mRNAs encoding one or more lysosomal and/or related proteins listed in Table 3;
thus, compositions of the invention may comprise an mRNA encoding a protein listed in Table 3 (or a homolog thereof) along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a protein chosen from the proteins listed in Table 3 (or a homolog thereof) along with other components set out herein.
183 Table 3. Lysosomal and Related Proteins a-fucosidase a-galactosidase a-glucosidase a-Iduronidase a-mannosidase a-N-acetylgalactosaminidase (a-galactosidase B) 13-galactosidase 13-glucuronidase 13-hexosaminidase 13-mannosidase 3¨hydroxy-3¨methylglutaryl¨CoA (HMG¨CoA) lyase 3¨methylcrotonyl¨CoA carboxylase 3 -0-sulfogalactosyl cerebroside sulfatase (arylsulfatase A) acetyl-CoA transferase acid alpha-glucosidase acid ceramidase acid lipase acid phosphatase acid sphingomyelinase alpha-galactosidase A
arylsulfatase A
beta-galactosidase beta-glucocerebrosidase beta-hexosaminidase biotinidase cathepsin A
cathepsin K

cystine transporter (cystinosin) cytosolic protein beta3A subunit of the adaptor protein-3 complex, AP3 formyl-Glycine generating enzyme (FGE) galactocerebrosidase galactose-1--phosphate uridyltransferase (GALT)
184 galactose 6-sulfate sulfatase (also known as N-acetylgalactosamine-6-sulfatase) glucocerebrosidase glucuronate sulfatase glucuronidase glycoprotein cleaving enzymes glycosaminoglycan cleaving enzymes glycosylasparaginase (aspartylglucosaminidase) Heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT, TMEM76) Heparan sulfatase hexosaminidase A lysosomal proteases methylmalonyl¨CoA mutase hyaluronidase Iduronate sulfatase lysosomal a-mannosidase Lysosomal p40 (C2orf18) Major facilitator superfamily domain containing 8 protein (MFSD8 or CLN7) N-acetylgalactosamine 4-sulfatase N-acetyl glucosamine 6-sulfatase N-acetyl glucosaminidase N-acetylglucosamine-l-phosphate transferase palmitoyl-protein thioesterase palmitoyl-protein thioesterase (CLN1) Saposin A (Sphingolipid activator protein A) Saposin B (Sphingolipid activator protein B) Saposin C (Sphingolipid activator protein C) Saposin D (Sphingolipid activator protein D) sialic acid transporter (sialin) sialidase Sialin sulfatase Transmembrane protein 74 (TMEM74) tripeptidyl-peptidase tripeptidyl-peptidase I (CLN2) UDP-N-acetylglucosamine- phosphotransferase
185 [0381] Information regarding lysosomal proteins is available from Lubke et al., "Proteomics of the Lysosome," Biochim Biophys Acta. (2009) 1793: 625-635. In some embodiments, the protein listed in Table 3 and encoded by mRNA in the compositions and methods of the invention is a human protein. Sequences of the listed proteins are also available for various animals, including various mammals and animals of veterinary or industrial interest as described above.
[0382] In some embodiments, the compositions and methods of the invention provide for the delivery of mRNA encoding a therapeutic protein (e.g., cytosolic, transmembrane or secreted) such as those listed in Table 4. In some embodiments, the compositions and methods of the invention provide for the delivery of an mRNA encoding a therapeutic protein useful in treating a disease or disorder (i.e., indication) listed in Table 4; thus, compositions of the invention may comprise an mRNA encoding a therapeutic protein listed or not listed in Table 4 (or a homolog thereof, as discussed below) along with other components set out herein for treating a disease or disorder (i.e., indication) listed in Table 4, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA
encoding a such a protein (or a homolog thereof, as discussed below) along with other components set out herein for treatment of a disease or disorder listed in Table 4.
Table 4. Exemplary Indications and Related Proteins Indication Therapeutic Protein 3-Methylcrotonyl-CoA carboxylase deficiency Methylcrotonoyl-CoA carboxylase 3-Methylglutaconic aciduria Methylglutaconyl-CoA hydratase Actinic keratosis Acute intermittent porphyria Porphobilinogen deaminase Acute lymphocytic leukemia Acute myeloid leukemia Addison's disease Adenosine deaminase deficiency Adenosine deaminase Adrenoleukodystrophy ABCD1 Adrenomyeloneuropathy AIDS/HIV
Alcohol use disorders Alkaptonuria Homogentis ate 1,2-dioxygenase Allergic asthma Anti-IgE mAb Allergies (dermatitis, rhinitis)
186 Alopecia areata Alpers' disease POLG
Alpers-Huttenlocher syndrome Alpha 1-antitrypsin deficiency Alpha 1 protease inhibitor Alpha-mannosidosis Alpha-D-mannosidase Alport syndrome Alzheimer's disease Amyloid light-chain amyloidosis Amyotrophic lateral sclerosis (ALS) Anemia Erythropoietin Aortic valve stenosis Argininemia Arginase Argininosuccinic acidemia Argininosuccinate lyase Arrhythmogenic right ventricular dysplasia Autism Autosomal dominant and recessive progressive external ophthalmoplegia with mitochondrial DNA
deletions Autosomal recessive polycystic kidney disease ARPKD
Bacterial infections Basal cell carcinoma Batten disease Battenin + others B-cell chronic lymphocytic leukemia Becker muscular dystrophy Dystrophin Beta-thalassemia Beta globin Binge eating disorder Bipolar disorder Bladder cancer Blepharospasm, Cervical dystonia, Chronic migraine, Botulinum toxin more Bronchiolitis obliterans Brugada syndrome Buerger's disease CACNB4-related Episodic Ataxia Type 2 Cancer and depression Cancer and sexual dysfunction Cancer in pregnancy Carbamylphosphate synthetase deficiency Carbamylphosphate synthetase Carcinoma of the gallbladder Cardiomyopathy (diabetic) Cardiomyopathy (hypertrophic) Carnitine uptake defect SLC22A5 Catecholaminergic polymorphic ventricular tachycardia CDKL5-related Atypical Rett Syndrome Celiac disease Cellulitis
187 Cerebrovascular disease Cervix uteri cancer Chronic fatigue syndrome Chronic graft versus host disease Chronic idiopathic urticaria Chronic immune thrombocytopenia Thrombopoietin Chronic kidney kisease Chronic liver disease Chronic lymphocytic leukemia Chronic myeloid leukemia Chronic pancreatitis Cirrhosis of the liver Citrullinemia, type I Argininosuccinate synthase Classic Rett Syndrome Classical galactosemia Galactose- 1-phosphate uridylyltransferase Clostridium difficile associated diarrhea Clotting disorders COAD/COPD
Cocaine addiction COL4A5-related disorders Cold contact urticaria Contraception, female Coronary artery diseases Corpus uteri cancer Corticobasal degeneration Crigler-Najjar syndrome UDP-glucuronosyltransferase Critical limb ischemia CTNS-related cystinosis Cutaneous lupus erythematosus Cutaneous neuroendocrine carcinoma (Merkel Cell) Cystic fibrosis CFTR
Cystic fibrosis Deoxyribonuclease I
Cystinosis Cystinosin Cystinuria SLC7A9 Dementia (Lewy body) Depression Diabetic foot infections Diabetic foot ulcer Diabetic peripheral neuropathy Diabetic ulcers Diarrhoeal diseases Diffuse large B-cell lymphoma DiGeorge syndrome Diverticulitis Drug use disorders Duchenne muscular dystrophy Dystrophin Dysarthria Dyskinesia (levodopa-induced)
188 Early-onset autosomal dominant Alzheimer's disease Eczema Ehlers-Danlos syndrome, type 1 EIF2B5-related childhood ataxia with central nervous system hypomyelination/vanishing white matter Eosinophilic esophagitis Epilepsy Erectile dysfunction Erythropoietic protoporphyria Ferrochelatase Esophageal carcinoma Essential tremor Fabry disease Alpha galactosidase Familial adenomatous polyposis APC
Familial chylomicronemia Lipoprotein lipase Familial dysbetalipoproteinemia Apolipoprotein E
Familial isolated dilated cardiomyopathy Familial mediterranean fever Pyrin (MEFV) Familial melanoma Female infertility Follicle stimulating hormone Female sexual dysfunction Fibromyalgia FMR1-related disorders Fracture healing Fragile X Premature Ovarian Failure Syndrome Fragile X syndrome FMRP
Fragile X-Associated Tremor/Ataxia Syndrome Friedreich's ataxia Frontotemporal dementia Fryns syndrome Galactocerebrosidase deficiencies GALE deficiency Galactose epimerase GALK deficiency Galactokinase GALT-related galactosemia Gastric cancer Gastroesophageal reflux disease Gaucher disease Glucocerebrosidase Gilbert syndrome UDP-glucuronosyltransferase Glioblastoma multiforme Glomerulonephritis Glutaric acidemia, type I Glutaryl-CoA dehydrogenase GM2 gangliosidosis HEXA, HEXB
Gout Urate oxidase Graft versus host disease Growth hormone deficiency Growth hormone 1 / Growth hormone 2
189 Head and neck cancer, Metastatic colorectal cancer Anti-EGFr mAb Hearing loss, adult onset Heart failure Hemachromatosis HFE protein Hemifacial spasm Hemolytic uremic syndrome Anti-complement factor C5 mAb Hemophilia A Factor VIII
Hemophilia A, Hemophilia B Factor VII
Hemophilia B Factor IX
Hepatitis B, Hepatitis C Interferon alpha HER2+ breast cancer, gastric cancer Anti-HER2 mAb Hereditary angioedema Cl esterase inhibitor Hereditary hemorrhagic telangiectasia Hereditary hemorrhagic telangiectasia (AT) Hereditary spherocytosis Hidradenitis suppurativa Homocystinuria Cystathionine beta- synthase Homozygous familial hypercholesterolemia LDL receptor Hunter syndrome (MPS II) Iduronate-2-sulfatase Huntington disease Huntingtin Hurler syndrome (MPS I) Alpha-L iduronidase Hydrolethalus Hyperalgesia Hyperbilirubinemia Hyperhidrosis Hyperlipidemia Hypermethioninemia Methionine adenosyltransferase Hyperoxaluria, type I Serine-pyruvate aminotransferase Hypertension Hyperuricemia Hyponatremia Hypoparathyroidism Parathyroid hormone Hypophosphatasia TNSALP
Idiopathic pulmonary fibrosis Iminoglycinuria Immunoglobulin deficiency Immunoglobulin Infection (adenovirus) Infection (anthrax prophylaxis) Infection (BK virus) Infection (Clostridium difficile prophylaxis) Infection (Dengue fever prophylaxis) Infection (Epstein-Barr virus) Infection (Hepatitis-D) Infection (Lyme disease prophylaxis) Infection (Smallpox virus) Infectious diseases vaccines Infectious antigen Inflammatory heart diseases Insomnia
190 Interstitial cystitis Iron-deficiency anaemia Irritable bowel disease Ischaemic heart disease Isovaleric aciduria Isovaleric acid CoA dehydrogenase deficiency Jansky-Bielschowsky disease Juvenile Batten disease Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) Juvenile rheumatoid arthritis TNF-alpha inhibitors Kennedy's disease (SBMA) Keratoconus Krabbe disease Galactocerebrosidase Leber's hereditary optic neuropathy NADH
dehydrogenase Leiomyosarcoma Lennox-Gastaut syndrome Lesch-Nyhan syndrome Hypoxanthine phosphoribosyltransferase 1 Leukaemia Li-Fraumeni syndrome TP53 Lipoma Liposarcoma Liver cancer Long-chain 3-0H acyl-CoA dehydrogenase deficiency Long-chain-3-hydroxyacyl-CoA
dehydrogenase Lower respiratory infections Lysosomal acid lipase deficiency Lysosomal acid lipase Macular degeneration Major depressive disorder Malignant fibrous histiocytoma Mantle cell lymphoma Maple syrup urine disease 3-methyl-2-oxobutanoate dehydrogenase Marfan syndrome FBN1 Maroteaux-Lamy syndrome (MPS VI) N-acetylgalactosamine 4-sulfatase Mastocytosis McArdle disease Muscle glycogen phosphorylase MECP2-related disorders MECP2-related Severe Neonatal Encephalopathy Medium-chain acyl-CoA dehydrogenase deficiency Acyl-CoA dehydrogenase Melanoma Anti-CTLA4 mAb Metachromatic leukodystrophy Arylsulfatase A
Metastatic colorectal cancer, NSCLC, others Anti-VEGF mAb Methylmalonyl-CoA mutase deficiency Methylmalonyl-CoA mutase Migraine Mitochondrial oxidative phosphorylation disorders Morquio syndrome, type A (MPS IVA) Galactose 6-sulfate sulfatase Morquio syndrome, type B (MPS IVB) Beta-galactosidase Mouth and oropharynx cancers Multiple carboxylase deficiency Biotin-methylcrotonoyl-CoA-carboxylase ligase Multiple myeloma Multiple sclerosis Anti-VLA-4 mAb
191 Multiple sclerosis Interferon beta Multiple system atrophy Myasthenia gravis Myelofibrosis Narcolepsy Neonatal bronchopulmonary dysplasia Neonatal infections Nephritis and nephrosis Neurofibromatosis, type 1 NF-1 Neuronal ceroid lipofuscinoses-related diseases Neutropenia G-CSF
Niemann Pick disease, type A / B SMPD1 Niemann Pick disease, type C NPC1 Niemann-Pick disease Type Cl Nocturia Non-alcoholic fatty liver disease Non-Hodgkin lymphoma Anti-CD20 mAb Non-small cell lung cancer Notch-3 related cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADAS IL) Obesity Ophthalmoparesis Opioid induced constipation Ornithine transcarbamylase deficiency Ornithine transcarbamylase Osteoarthritis Osteopetrosis Osteoporosis Anti-RANKL mAb Ovarian cancer Paget disease of bone Sequestosome 1 Pain Pancreatic carcinoma Panic disorder Parkinson disease Paroxysmal nocturnal hemoglobinuria Anti-complement factor C5 Mab Pediculosis capitis (head lice) Pelizaeus-Merzbacher disease Pemphigus vulgaris Peptic ulcer disease Peripheral neuropathy Peyronie's disease Phenylketonuria Phenylalanine hydroxylase Pneumococcal infection prophylaxis POLG-related sensory ataxic neuropathy Polycystic kidney disease Polycystic ovary syndrome Polycythaemia vera Polymerase G-related disorders
192 Polymorphous light eruption Pompe disease Alpha glucosidase Porphyria cutanea tarda Uroporphyrinogen decarboxylase Post herpetic neuralgia Post-organ transplant Pouchitis PPM-X Syndrome Prader-Willi syndrome Preeclampsia Premature ejaculation Prematurity and low birth weight Primary ciliary dyskinesia Primary glomerular diseases Primary humoral immune deficiencies (e.g., CVID) Immunoglobulin Proctitis Progressive multifocal leukoencephalopathy Progressive supranuclear palsy Propionic acidemia Propionyl-CoA carboxylase Prostate cancer Psoriasis Anti-IL-12 & IL-23 mAb Psoriatic arthritis TNF-alpha inhibitors Pulmonary arterial hypertension Pulmonary arterial hypertension Raynaud's phenomenon Refractive errors Renal cell carcinoma Restless leg syndrome Retinitis pigmentosa Rheumatic heart disease Rheumatoid arthritis Anti-interleukin-6 (IL-6) mAb Rheumatoid arthritis T-cell costimulation blocker Rheumatoid arthritis TNF-alpha inhibitor Romano-Ward syndrome Rosacea Sanfilippo syndrome, type A (MPS IIIA) Heparan N-sulfatase Sanfilippo syndrome, type B (MPS IIIB) N-acetyl-alpha-D-glucosaminidase Santavuori-Haltia disease Schizophrenia Schnitzler syndrome Scleroderma SCN1B-related seizure disorders Short-chain acyl-CoA dehydrogenase deficiency Butyryl-CoA dehydrogenase Sickle cell disease Hemoglobin SLC3A1-related disorders Small cell lung cancer SMN-1-related spinal muscular atrophy (SMA)
193 Spinal muscular atrophy Survival motor neuron protein Squamous cell carcinoma of head and neck Stickler syndrome Stomach cancer Stroke prophylaxis Synovial sarcoma Systemic lupus erythematosus Anti-BAFF
Systemic sclerosis Tetrahydrobiopterin-deficient hyperphenylalaninemia Tetrahydrobiopterin Thromboangiitis obliterans Thrombotic disorders Thyroid cancer TPP1 deficiencies Trachea, bronchus, lung cancers Tricuspid atresia TSC2-related tuberous sclerosis Type 2 diabetes mellitus Glucagon-like peptide 1 (GLP-1) agonist Type 2 diabetes mellitus Insulin Tyrosinemia, type I Fumarylacetoacetase Ulcerative colitis Uterine fibroids Varicose veins Venous thromboembolism Very long-chain acyl-CoA dehydrogenase deficiency Long-chain-acyl-CoA
dehydrogenase von Gierke's disease Glucose-6-phosphatase Von Hippel-Lindau disease pVHL
Wegener granulomatosis Wilson disease Wilson disease protein X-Linked adrenal hypoplasia X-linked adrenoleukodystrophy X-linked agammaglobulinemia Bruton's tyrosine kinase [0383] In some embodiments, the present invention is used to prevent, treat and/or cure a subject affected with a disease or disorder listed or associated with the proteins listed in Tables 1, 2, 3 or 4. In some embodiments, an mRNA encodes one or more of argininosuccinate synthetase (ASS1), Factor IX, survival motor neuron 1 (SMN1), or phenylalanine hydroxylase Synthesis of mR1VA
[0384] mRNAs according to the present invention may be synthesized according to any of a variety of known methods. For example, mRNAs according to the present invention may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or
194 circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA
polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor. The exact conditions will vary according to the specific application.
[0385] In some embodiments, for the preparation of mRNA according to the invention, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter, for example a T3, T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and a termination signal.
[0386] Desired mRNA sequence(s) according to the invention may be determined and incorporated into a DNA template using standard methods. For example, starting from a desired amino acid sequence (e.g., an enzyme sequence), a virtual reverse translation is carried out based on the degenerated genetic code. Optimization algorithms may then be used for selection of suitable codons. Typically, the G/C content can be optimized to achieve the highest possible G/C content on one hand, taking into the best possible account the frequency of the tRNAs according to codon usage on the other hand. The optimized RNA sequence can be established and displayed, for example, with the aid of an appropriate display device and compared with the original (wild-type) sequence. A secondary structure can also be analyzed to calculate stabilizing and destabilizing properties or, respectively, regions of the RNA.
Modified mRNA
[0387] In some embodiments, mRNA according to the present invention may be synthesized as unmodified or modified mRNA. Typically, mRNAs are modified to enhance stability. Modifications of mRNA can include, for example, modifications of the nucleotides of the RNA. An modified mRNA according to the invention can thus include, for example, backbone modifications, sugar modifications or base modifications. In some embodiments, mRNAs may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g. 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-
195 cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethy1-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methy1-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethy1-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, .beta.-D-mannosyl-queosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. The preparation of such analogues is known to a person skilled in the art e.g. from the U.S. Pat. No.
4,373,071, U.S. Pat.
No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No.
4,500,707, U.S.
Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S.
Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. Nos. 5,262,530 and 5,700,642, the disclosures of which are incorporated by reference in their entirety.
[0388] In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) may contain RNA backbone modifications. Typically, a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g. cytidine 5'-0-(1-thiophosphate)), boranophosphates, positively charged guanidinium groups etc., which means by replacing the phosphodiester linkage by other anionic, cationic or neutral groups.
[0389] In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) may contain sugar modifications. A typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2'-deoxy-2'-fluoro-oligoribonucleotide (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotide (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-0-alkyloligoribonucleotide, 2'-deoxy-2'-C-alkyloligoribonucleotide (2'-0-methylcytidine 5'-
196 triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyloligoribonucleotide, and isomers thereof (2'-aracytidine 5'-triphosphate, 2'-arauridine 5'-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).
[0390] In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) may contain modifications of the bases of the nucleotides (base modifications). A modified nucleotide which contains a base modification is also called a base-modified nucleotide.
Exemples of such base-modified nucleotides include, but are not limited to, 2-amino-6-chloropurine riboside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate, 6-azacytidine 5'-triphosphate, 6-azauridine 5'-triphosphate, 6-chloropurine riboside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole riboside 5'-triphosphate, Nl-methyladenosine 5'-triphosphate, N1-methylguanosine 5'-triphosphate, N6-methyladenosine 5'-triphosphate, 06-methylguanosine 5'-triphosphate, pseudouridine 5'-triphosphate, puromycin 5'-triphosphate or xanthosine 5'-triphosphate.
Cap Structure [0391] Typically, mRNA synthesis includes the addition of a "cap" on the N-terminal (5') end, and a "tail" on the C-terminal (3') end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a "tail" serves to protect the mRNA from exonuclease degradation.
[0392] Thus, in some embodiments, mRNAs (e.g., enzyme encoding mRNAs) include a 5' cap structure. A 5' cap is typically added as follows: first, an RNA
terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5'5'5 triphosphate linkage; and the 7-nitrogen of guanine is
197 then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G.
[0393] In some embodiments, naturally occurring cap structures comprise a 7-methyl guanosine that is linked via a triphosphate bridge to the 5'-end of the first transcribed nucleotide, resulting in a dinucleotide cap of m7G(5')ppp(5')N, where N is any nucleoside.
In vivo, the cap is added enzymatically. The cap is added in the nucleus and is catalyzed by the enzyme guanylyl transferase. The addition of the cap to the 5' terminal end of RNA occurs immediately after initiation of transcription. The terminal nucleoside is typically a guanosine, and is in the reverse orientation to all the other nucleotides, i.e., G(5')ppp(5')GpNpNp.
[0394] A common cap for mRNA produced by in vitro transcription is m7G(5')ppp(5')G, which has been used as the dinucleotide cap in transcription with T7 or SP6 RNA polymerase in vitro to obtain RNAs having a cap structure in their 5'-termini. The prevailing method for the in vitro synthesis of capped mRNA employs a pre-formed dinucleotide of the form m7G(5')ppp(5')G ("m7GpppG") as an initiator of transcription.
[0395] To date, a usual form of a synthetic dinucleotide cap used in in vitro translation experiments is the Anti-Reverse Cap Analog ("ARCA") or modified ARCA, which is generally a modified cap analog in which the 2' or 3' OH group is replaced with -OCH3.
[0396] Additional cap analogs include, but are not limited to, chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog (e.g., m2'7GpppG), trimethylated cap analog (e.g., m2'2'7GpppG), dimethylated symmetrical cap analogs (e.g., m7Gpppm7G), or anti reverse cap analogs (e.g., ARCA; m7,2'OmeGpppG, m72'dGpppG, m7'3'OmeGpppG, m7'3'dGpppG
and their tetraphosphate derivatives) (see, e.g., Jemielity, J. et al., "Novel 'anti-reverse' cap analogs with superior translational properties", RNA, 9: 1108-1122 (2003)).
[0397] In some embodiments, a suitable cap is a 7-methyl guanylate ("m7G") linked via a triphosphate bridge to the 5'-end of the first transcribed nucleotide, resulting in m7G(5')ppp(5')N, where N is any nucleoside. A preferred embodiment of a m7G cap utilized in embodiments of the invention is m7G(5')ppp(5')G.
198 [0398] In some embodiments, the cap is a Cap structure. Cap structures lack a 2'-0-methyl residue of the ribose attached to bases 1 and 2. In some embodiments, the cap is a Capl structure. Capl structures have a 2'-0-methyl residue at base 2. In some embodiments, the cap is a Cap2 structure. Cap2 structures have a 2'-0-methyl residue attached to both bases 2 and 3.
[0399] A variety of m7G cap analogs are known in the art, many of which are commercially available. These include the m7GpppG described above, as well as the ARCA 3'-OCH3 and 2'-OCH3 cap analogs (Jemielity, J. et al., RNA, 9: 1108-1122 (2003)).
Additional cap analogs for use in embodiments of the invention include N7-benzylated dinucleoside tetraphosphate analogs (described in Grudzien, E. et al., RNA, 10: 1479-1487 (2004)), phosphorothioate cap analogs (described in Grudzien-Nogalska, E., et al., RNA, 13: 1745-1755 (2007)), and cap analogs (including biotinylated cap analogs) described in U.S. Patent Nos.
8,093,367 and 8,304,529, incorporated by reference herein.
Tail Structure [0400] Typically, the presence of a "tail" serves to protect the mRNA
from exonuclease degradation. The poly A tail is thought to stabilize natural messengers and synthetic sense RNA.
Therefore, in certain embodiments a long poly A tail can be added to an mRNA
molecule thus rendering the RNA more stable. Poly A tails can be added using a variety of art-recognized techniques. For example, long poly A tails can be added to synthetic or in vitro transcribed RNA
using poly A polymerase (Yokoe, et at. Nature Biotechnology. 1996; 14: 1252-1256). A
transcription vector can also encode long poly A tails. In addition, poly A
tails can be added by transcription directly from PCR products. Poly A may also be ligated to the 3' end of a sense RNA with RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).
[0401] In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) include a 3' poly(A) tail structure. Typically, the length of the poly A tail can be at least about 10, 50, 100, 200, 300, 400 at least 500 nucleotides (SEQ ID NO: 12). In some embodiments, a poly-A tail on the 3' terminus of mRNA typically includes about 10 to 300 adenosine nucleotides (SEQ ID NO:
13) (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine
199 nucleotides). In some embodiments, mRNAs include a 3' poly(C) tail structure.
A suitable poly-C tail on the 3' terminus of mRNA typically include about 10 to 200 cytosine nucleotides (SEQ ID NO: 14) (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail may be added to the poly-A tail or may substitute the poly-A tail.
[0402] In some embodiments, the length of the poly A or poly C tail is adjusted to control the stability of a modified sense mRNA molecule of the invention and, thus, the transcription of protein. For example, since the length of the poly A tail can influence the half-life of a sense mRNA molecule, the length of the poly A tail can be adjusted to modify the level of resistance of the mRNA to nucleases and thereby control the time course of polynucleotide expression and/or polypeptide production in a target cell.
5' and 3' Untranslated Region [0403] In some embodiments, mRNAs include a 5' and/or 3' untranslated region. In some embodiments, a 5' untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element. In some embodiments, a 5' untranslated region may be between about 50 and 500 nucleotides in length.
[0404] In some embodiments, a 3' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3' untranslated region may be between 50 and 500 nucleotides in length or longer.
[0405] Exemplary 3' and/or 5' UTR sequences can be derived from mRNA
molecules which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the sense mRNA molecule. For example, a 5' UTR
sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof to improve the nuclease resistance and/or improve the half-life of the polynucleotide. Also contemplated is the inclusion of a sequence encoding human growth hormone (hGH), or a fragment thereof to the 3' end or untranslated region of the polynucleotide (e.g., mRNA) to further stabilize the polynucleotide. Generally, these modifications improve the stability and/or
200 pharmacokinetic properties (e.g., half-life) of the polynucleotide relative to their unmodified counterparts, and include, for example modifications made to improve such polynucleotides' resistance to in vivo nuclease digestion.
[0406] According to various embodiments, any size mRNA may be encapsulated by provided liposomes. In some embodiments, the provided liposomes may encapsulate mRNA of greater than about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb in length.
Formation of Liposomes [0407] The liposomes for use in provided compositions can be prepared by various techniques which are presently known in the art. For example, multilamellar vesicles (MLV) may be prepared according to conventional techniques, such as by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then added to the vessel with a vortexing motion which results in the formation of MLVs. Uni-lamellar vesicles (ULV) can then be formed by homogenization, sonication or extrusion of the multi-lamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.
[0408] In certain embodiments, provided compositions comprise a liposome wherein the mRNA is associated on both the surface of the liposome and encapsulated within the same liposome. For example, during preparation of the compositions of the present invention, cationic liposomes may associate with the mRNA through electrostatic interactions. For example, during preparation of the compositions of the present invention, cationic liposomes may associate with the mRNA through electrostatic interactions.
[0409] In some embodiments, the compositions and methods of the invention comprise mRNA encapsulated in a liposome. In some embodiments, the one or more mRNA
species may be encapsulated in the same liposome. In some embodiments, the one or more mRNA species may be encapsulated in different liposomes. In some embodiments, the mRNA is encapsulated in one or more liposomes, which differ in their lipid composition, molar ratio of lipid components, size, charge (Zeta potential), targeting ligands and/or combinations thereof In
201 some embodiments, the one or more liposome may have a different composition of cationic lipids, neutral lipid, PEG-modified lipid and/or combinations thereof. In some embodiments the one or more lipisomes may have a different molar ratio of cationic lipid, neutral lipid, cholesterol and PEG-modified lipid used to create the liposome.
[0410] The process of incorporation of a desired mRNA into a liposome is often referred to as "loading". Exemplary methods are described in Lasic, et al., FEBS Lett., 312: 255-258, 1992, which is incorporated herein by reference. The liposome-incorporated nucleic acids may be completely or partially located in the interior space of the liposome, within the bilayer membrane of the liposome, or associated with the exterior surface of the liposome membrane.
The incorporation of a nucleic acid into liposomes is also referred to herein as "encapsulation"
wherein the nucleic acid is entirely contained within the interior space of the liposome. The purpose of incorporating a mRNA into a transfer vehicle, such as a liposome, is often to protect the nucleic acid from an environment which may contain enzymes or chemicals that degrade nucleic acids and/or systems or receptors that cause the rapid excretion of the nucleic acids.
Accordingly, in some embodiments, a suitable delivery vehicle is capable of enhancing the stability of the mRNA contained therein and/or facilitate the delivery of mRNA
to the target cell or tissue.
Liposome Size [0411] Suitable liposomes in accordance with the present invention may be made in various sizes. In some embodiments, provided liposomes may be made smaller than previously known mRNA encapsulating liposomes. In some embodiments, decreased size of liposomes is associated with more efficient delivery of mRNA. Selection of an appropriate liposome size may take into consideration the site of the target cell or tissue and to some extent the application for which the liposome is being made.
[0412] In some embodiments, an appropriate size of liposome is selected to facilitate systemic distribution of antibody encoded by the mRNA. In some embodiments, it may be desirable to limit transfection of the mRNA to certain cells or tissues. For example, to target hepatocytes a liposome may be sized such that its dimensions are smaller than the fenestrations
202 of the endothelial layer lining hepatic sinusoids in the liver; in such cases the liposome could readily penetrate such endothelial fenestrations to reach the target hepatocytes.
[0413] Alternatively or additionally, a liposome may be sized such that the dimensions of the liposome are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues. For example, a liposome may be sized such that its dimensions are larger than the fenestrations of the endothelial layer lining hepatic sinusoids to thereby limit distribution of the liposomes to hepatocytes.
[0414] In some embodiments, the size of a liposome is determined by the length of the largest diameter of the lipososme particle. In some embodiments, a suitable liposome has a size of or less than about 500nm, 450 nm, 400nm, 350 nm, 300nm, 250 nm, 200 nm, 150 nm, 125 nm, 110 nm, 100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, or 50 nm. In some embodiments, a suitable liposome has a size no greater than about 250 nm (e.g., no greater than about 225 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, or 50 nm). In some embodiments, a suitable liposome has a size ranging from about 10 - 250 nm (e.g., ranging from about 10 -225 nm, 10 -200 nm, 10- 175 nm, 10- 150 nm, 10- 125 nm, 10- 100 nm, 10 -75 nm, or 10 - 50 nm). In some embodiments, a suitable liposome has a size ranging from about 100 - 250 nm (e.g., ranging from about 100 - 225 nm, 100 - 200 nm, 100 - 175 nm, 100 - 150 nm). In some embodiments, a suitable liposome has a size ranging from about 10 - 100 nm (e.g., ranging from about 10 - 90 nm, 10 - 80 nm, 10 - 70 nm, 10 - 60 nm, or 10 - 50 nm).
[0415] A variety of alternative methods known in the art are available for sizing of a population of liposomes. One such sizing method is described in U.S. Pat. No.
4,737,323, incorporated herein by reference. Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small ULV less than about 0.05 microns in diameter. Homogenization is another method that relies on shearing energy to fragment large liposomes into smaller ones. In a typical homogenization procedure, MLV are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed. The size of the liposomes may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann.
Rev. Biophys.
Bioeng., 10:421-150 (1981), incorporated herein by reference. Average liposome diameter may
203 be reduced by sonication of formed liposomes. Intermittent sonication cycles may be alternated with QELS assessment to guide efficient liposome synthesis.
Pharmaceutical Compositions [0416] To facilitate expression of mRNA in vivo, delivery vehicles such as liposomes can be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or in pharmacological compositions where it is mixed with suitable excipients. Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.
[0417] Provided liposomally-encapsulated or associated mRNAs, and compositions containing the same, may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subject's age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art. The "effective amount" for the purposes herein may be determined by such relevant considerations as are known to those of ordinary skill in experimental clinical research, pharmacological, clinical and medical arts.
In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indicators as are selected as appropriate measures of disease progress, regression or improvement by those of skill in the art. For example, a suitable amount and dosing regimen is one that causes at least transient protein (e.g., enzyme) production.
[0418] Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration;
parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, and/or intranasal administration.
[0419] Alternately or additionally, liposomally encapsulated mRNAs and compositions of the invention may be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a targeted tissue, preferably in a
204 sustained release formulation. Local delivery can be affected in various ways, depending on the tissue to be targeted. For example, aerosols containing compositions of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); compositions of the present invention can be injected into the site of injury, disease manifestation, or pain, for example; compositions can be provided in lozenges for oral, tracheal, or esophageal application; can be supplied in liquid, tablet or capsule form for administration to the stomach or intestines, can be supplied in suppository form for rectal or vaginal application; or can even be delivered to the eye by use of creams, drops, or even injection. Formulations containing provided compositions complexed with therapeutic molecules or ligands can even be surgically administered, for example in association with a polymer or other structure or substance that can allow the compositions to diffuse from the site of implantation to surrounding cells. Alternatively, they can be applied surgically without the use of polymers or supports.
[0420] In some embodiments, provided liposomes and/or compositions are formulated such that they are suitable for extended-release of the mRNA contained therein. Such extended-release compositions may be conveniently administered to a subject at extended dosing intervals.
For example, in one embodiment, the compositions of the present invention are administered to a subject twice day, daily or every other day. In a preferred embodiment, the compositions of the present invention are administered to a subject twice a week, once a week, every ten days, every two weeks, every three weeks, or more preferably every four weeks, once a month, every six weeks, every eight weeks, every other month, every three months, every four months, every six months, every eight months, every nine months or annually. Also contemplated are compositions and liposomes which are formulated for depot administration (e.g., intramuscularly, subcutaneously, intravitreally) to either deliver or release a mRNA over extended periods of time. Preferably, the extended-release means employed are combined with modifications made to the mRNA to enhance stability.
[0421] Also contemplated herein are lyophilized pharmaceutical compositions comprising one or more of the liposomes disclosed herein and related methods for the use of such compositions as disclosed for example, in United States Provisional Application No.
61/494,882, filed June 8, 2011, the teachings of which are incorporated herein by reference in their entirety. For example, lyophilized pharmaceutical compositions according to the invention
205 may be reconstituted prior to administration or can be reconstituted in vivo.
For example, a lyophilized pharmaceutical composition can be formulated in an appropriate dosage form (e.g., an intradermal dosage form such as a disk, rod or membrane) and administered such that the dosage form is rehydrated over time in vivo by the individual's bodily fluids.
[0422] Provided liposomes and compositions may be administered to any desired tissue.
In some embodiments, the mRNA delivered by provided liposomes or compositions is expressed in the tissue in which the liposomes and/or compositions were administered. In some embodiments, the mRNA delivered is expressed in a tissue different from the tissue in which the liposomes and/or compositions were administered Exemplary tissues in which delivered mRNA
may be delivered and/or expressed include, but are not limited to the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and/or cerebrospinal fluid.
[0423] According to various embodiments, the timing of expression of delivered mRNAs can be tuned to suit a particular medical need. In some embodiments, the expression of the protein encoded by delivered mRNA is detectable 1, 2, 3, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and/or 72 hours in serum or target tissues after a single administration of provided liposomes or compositions. In some embodiments, the expression of the protein encoded by the mRNA is detectable 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and/or 7 days in serum or target tissues after a single administration of provided liposomes or compositions. In some embodiments, the expression of the protein encoded by the mRNA is detectable 1 week, 2 weeks, 3 weeks, and/or 4 weeks in serum or target tissues after a single administration of provided liposomes or compositions. In some embodiments, the expression of the protein encoded by the mRNA is detectable after a month or longer after a single administration of provided liposomes or compositions.
[0424] The present invention can be used to deliver mRNA at various doses. In some embodiments, an mRNA is administered at a dose ranging from about 0.1 - 5.0 mg /kg body weight, for example about 0.1 - 4.5, 0.1 - 4.0, 0.1 - 3.5, 0.1 - 3.0, 0.1 -2.5, 0.1 - 2.0, 0.1- 1.5, 0.1 - 1.0, 0.1 - 0.5, 0.1 - 0.3, 0.3 - 5.0, 0.3 - 4.5, 0.3 -4.0, 0.3 -3.5, 0.3 -3.0, 0.3 -2.5, 0.3 -2.0, 0.3 - 1.5, 0.3 - 1.0, 0.3 - 0.5, 0.5 - 5.0, 0.5-4.5, 0.5 -4.0, 0.5 - 3.5, 0.5 - 3.0, 0.5 -2.5, 0.5 -2.0, 0.5 - 1.5, or 0.5 - 1.0 mg/kg body weight. In some embodiments, an mRNA
is administered
206 at a dose of or less than about 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.8, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mg/kg body weight.
EXAMPLES
[0425] While certain compounds, compositions and methods of the present invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the invention and are not intended to limit the same.
Example 1. Exemplary Liposome Formulations for mRNA Delivery and Expression [0426] This example provides exemplary liposome formulations incorporating the cationic lipids described in this application, for example, cKK-E12, for effective delivery and expression of mRNA encoding therapeutic proteins in vivo.
Lipid Materials [0427] In general, the formulations described herein are based on a multi-component lipid mixture of varying ratios employing one or more cationic lipids, one or more helper lipids (e.g., non-cationic lipids and/or cholesterol-based lipids), and one or more PEGylated lipids designed to encapsulate various nucleic acid-based materials. As a non-limiting example, cKK-E12 (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione) is used in various formulations described herein. Exemplary helper lipids include one or more of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), cholesterol, etc. Exemplary PEGylated lipids include a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length, for example, PEG-2K. As non-limiting examples, liposome formulations used in various examples described herein include cKK-E12, DOPE, cholesterol and DMG-PEG2K at various
207 ratios. For example, in some cases, the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:30:20:10 by weight. In other cases, the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:32:25:3 by weight. Unless otherwise specified, the below Examples include a mixture in the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K of approximately 40:30:25:5 by weight.
Messenger RNA Material [0428] The formulations described herein may be used to deliver any mRNA, in particular, therapeutic mRNA. As used herein, a therapeutic mRNA refers to an mRNA that encodes a therapeutic protein. The formulations described herein can also be used to deliver any modified or unmodified mRNA, or mRNA with naturally occurring sequences or codon-optimized.
[0429] As non-limiting examples, human Factor IX (FIX), codon-optimized Firefly Luciferase (FFL), codon-optimized human argininosuccinate synthetase (ASS1) messenger RNA, codon-optimized human Survival of Motor Neuron l(SMN) mRNA were synthesized by in vitro transcription from a plasmid DNA template encoding the gene, which was followed by the addition of a 5' cap structure (Cap 1) (Fechter, P.; Brownlee, G.G.
"Recognition of mRNA
cap structures by viral and cellular proteins" J. Gen. Virology 2005, 86, 1239-1249) and a 3' poly(A) tail of, e.g., approximately 250 nucleotides in length (SEQ ID NO: 15) as determined by gel electrophoresis. Typically, 5' and 3' untranslated regions (UTR) are present in each mRNA
product and are represented as X and Y, respectively. Example 5' and 3' UTR
sequences are described below. The exemplary sequences of FIX, ASS1, and FFL mRNA used in the examples herein are listed below. Also shown are the 5' and 3' UTR sequences.
Human Factor IX (FIX) mRNA:
XAUGCAGCGCGUGAACAUGAUCAUGGCAGAAUCACCAGGCCUCAUCACCAUCUGC
CUUUUAGGAUAUCUACUCAGUGCUGAAUGUACAGUUUUUCUUGAUCAUGAAAAC
GCCAACAAAAUUCUGAGGCGGAGAAGGAGGUAUAAUUCAGGUAAAUUGGAAGAG
UUUGUUCAAGGGAACCUUGAGAGAGAAUGUAUGGAAGAAAAGUGUAGUUUUGAA
GAAGCACGAGAAGUUUUUGAAAACACUGAAAGAACAACUGAAUUUUGGAAGCAG
UAUGUUGAUGGAGAUCAGUGUGAGUCCAAUCCAUGUUUAAAUGGCGGCAGUUGC
AAGGAUGACAUUAAUUCCUAUGAAUGUUGGUGUCCCUUUGGAUUUGAAGGAAAG
AACUGUGAAUUAGAUGUAACAUGUAACAUUAAGAAUGGCAGAUGCGAGCAGUUU
208 UGUAAAAAUAGUGCUGAUAACAAGGUGGUUUGCUCCUGUACUGAGGGAUAUCGA
CUUGCAGAAAACCAGAAGUCCUGUGAACCAGCAGUGCCAUUUCCAUGUGGAAGA
GUUUCUGUUUCACAAACUUCUAAGCUCACCCGUGCUGAGGCUGUUUUUCCUGAUG
UGGACUAUGUAAAUUCUACUGAAGCUGAAACCAUUUUGGAUAACAUCACUCAAA
GCACCCAAUCAUUUAAUGACUUCACUCGGGUUGUUGGUGGAGAAGAUGCCAAAC
CAGGUCAAUUCCCUUGGCAGGUUGUUUUGAAUGGUAAAGUUGAUGCAUUCUGUG
GAGGCUCUAUCGUUAAUGAAAAAUGGAUUGUAACUGCUGCCCACUGUGUUGAAA
CUGGUGUUAAAAUUACAGUUGUCGCAGGUGAACAUAAUAUUGAGGAGACAGAAC
AUACAGAGCAAAAGCGAAAUGUGAUUCGAAUUAUUCCUCACCACAACUACAAUG
CAGCUAUUAAUAAGUACAAC CAUGACAUUGCCCUUCUGGAACUGGAC GAACC CUU
AGUGCUAAACAGCUACGUUACACCUAUUUGCAUUGCUGACAAGGAAUACACGAA
CAUCUUCCUCAAAUUUGGAUCUGGCUAUGUAAGUGGCUGGGGAAGAGUCUUCCA
CAAAGGGAGAUCAGCUUUAGUUCUUCAGUAC CUUAGAGUUC CAC UUGUUGAC CG
AGCCACAUGUCUUCGAUCUACAAAGUUCACCAUCUAUAACAACAUGUUCUGUGCU
GGCUUCCAUGAAGGAGGUAGAGAUUCAUGUCAAGGAGAUAGUGGGGGACCCCAU
GUUACUGAAGUGGAAGGGACCAGUUUCUUAACUGGAAUUAUUAGCUGGGGUGAA
GAGUGUGCAAUGAAAGGCAAAUAUGGAAUAUAUACCAAGGUAUCCCGGUAUGUC
AACUGGAUUAAGGAAAAAACAAAGCUCACUUAAY (SEQ ID NO.: 1) Codon-Optimized Human Argininosuccinate Synthetase (ASS]) mRNA:
XAUGAGCAGCAAGGGCAGCGUGGUGCUGGCCUACAGCGGCGGCCUGGACACCAGC
UGCAUCCUGGUGUGGCUGAAGGAGCAGGGCUACGACGUGAUCGCCUACCUGGCCA
ACAUCGGC CAGAAGGAGGACUUC GAGGAGGC CC GCAAGAAG GC C CUGAAGCUGGG
C GC CAAGAAGGUGUUCAUCGAGGAC GUGAGC C GC GAGUUC GUGGAGGAGUUC AU
CUGGCCCGCCAUCCAGAGCAGCGCCCUGUACGAGGACCGCUACCUGCUGGGCACC
AGCCUGGCCCGCCCCUGCAUCGCCCGCAAGCAGGUGGAGAUCGCCCAGCGCGAGG
GCGCCAAGUACGUGAGCCACGGCGCCACCGGCAAGGGCAACGACCAGGUGCGCUU
CGAGCUGAGCUGCUACAGC CUG GC CC CC CAGAUCAAGGUGAUC GC CCC CUGGC GC
AUGCC CGAGUUCUACAACCGCUUCAAGGGCC GCAAC GAC CUGAUGGAGUAC GC CA
AGCAGCACGGCAUCCCCAUCCCCGUGACCCCCAAGAACCCCUGGAGCAUGGACGA
GAAC CUGAUGCACAUCAGCUAC GAGGC CGGCAUCCUGGAGAAC C C CAAGAAC C AG
GCCCCCCCCGGCCUGUACACCAAGACCCAGGACCCCGCCAAGGCCCCCAACACCCC
C GA CAUC CUGGAGAUC GAGUUCAAGAAGGGC GUGC C C GUGAAGGUGAC C AAC GU
GAAGGAC GGCAC CAC C CAC CAGAC CAGC CUGGAGCUGUUCAUGUAC CUGAAC GAG
GUGGC CGGCAAGCAC GGCGUGGGC CGCAUC GACAUC GUGGAGAAC CGCUUCAUC G
GCAUGAAGAGCCGCGGCAUCUACGAGACCCCCGCCGGCACCAUCCUGUACCACGC
C C AC CUGGACAUC GAGGCCUUCAC CAUGGAC C GC GAG GUGC GC AAGAUCAA GCAG
GGCCUGGGCCUGAAGUUCGCCGAGCUGGUGUACACCGGCUUCUGGCACAGCCCCG
AGUGCGAGUUCGUGCGCCACUGCAUCGCCAAGAGCCAGGAGCGCGUGGAGGGCAA
GGUGCAGGUGAGCGUGCUGAAGGGC CAGGUGUACAUC CUGGGC C GC GAGAGC C C C
CUGA GC C UGUACAAC GAGGAGCUGGUGAGCAUGAACGUGCAGGGC GACUAC GAG
CC CAC CGAC GC CAC CGGCUUCAUCAACAUCAACAGCCUGC GC CUGAAGGAGUAC C
ACCGCCUGCAGAGCAAGGUGACCGCCAAGUGAY (SEQ ID NO.: 2)
209 Codon-Optimized Firefly Luciferase ('EEL,) mRNA:
XAUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCACUCGAA
GACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCCUGGUGC
CC GGCAC CAUCGC CUUUACCGACGCAC AUAUC GAGGUGGAC AUUAC CUACGC C GA
GUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAA
UACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCC
GUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACA
ACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGU
GAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAU
ACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUG
UAC AC CUUC GUGACUUCC CAUUUGCCAC CCGGCUUCAAC GAGUAC GACUUC GUGC
CC GAGAGCUUC GAC CGGGAC AAAAC CAUC GCCCUGAUC AUGAACAGUAGUGGCAG
UACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUC
AGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCC
UCAGC GUGGUGC CAUUUCAC CAC GGCUUC GGCAUGUUC AC C AC GCUGGGCUACUU
GAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUU
GCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUU
AGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACG
AGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAA
ACGCUUCCACCUACCAGGCAUCCGCCAGGGCUACGGCCUGACAGAAACAACCAGC
GCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGG
UGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUG
UGAAC CAGC GCGGC GAGCUGUGCGUCC GUGGC CCCAUGAUC AUGAGC GGCUAC GU
UAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGC
GGC GACAUC GC CUACUGGGAC GAGGAC GAGC ACUUCUUCAUC GUGGAC C GGCUGA
AGAGCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAU
CCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGAC
GAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGA
CCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCU
GCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUU
GGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUC
GCCGUGUAAY (SEQ ID NO.: 3) Codon-Optimized Human Survival of Motor Neuron 1(SMN) mRNA:
XAUGGCCAUGAGCAGCGGAGGCAGCGGCGGAGGAGUGCCCGAGCAGGAGGACAG
CGUGCUGUUCAGGAGAGGCACCGGCCAGAGCGAUGACAGCGAUAUCUGGGACGA
UAC C GCUCUGAUC AAGGC CUAC GAC AAGGC C GUGGC CAGCUUCAAGC AC GC C CUG
AAAAACGGCGACAUCUGCGAGACCAGCGGCAAGCCCAAGACAACCCCCAAGAGAA
AGC CCGC CAAGAAGAAUAAGAGC CAGAAAAAGAACAC CGC CGC CAGCCUGCAG CA
GUGGAAGGUGGGCGACAAGUGCAGCGCCAUCUGGAGCGAGGACGGCUGCAUCUA
CC CCGC CAC CAUC GCCAG CAUC GACUUC AAGAGAGAGACCUGCGUGGUC GUGUAC
ACCGGCUACGGCAACAGAGAGGAGCAGAACCUGAGCGACCUGCUGAGCCCCAUUU
GUGAGGUGGCCAAUAACAUCGAACAGAACGCCCAGGAGAACGAGAAUGAAAGCC
AGGUGAGCACCGACGAGAGCGAGAACAGCAGAUCUCCUGGCAACAAGAGCGACAA
CAUCAAGCCUAAGUCUGCCCCUUGGAACAGCUUCCUGCCCCCUCCUCCACCCAUG
210 CC C GGAC C CAGACUGGGAC C C GGAAAACCUGGCCUGAAGUUCAAC GGAC CAC CUC
CCCCUCCACCUCCUCCCCCACCUCAUCUCCUGAGCUGCUGGCUGCCACCCUUCCCC
AGCGGACCCCCUAUCAUCCCACCACCCCCUCCCAUCUGCCCCGACAGCCUGGACGA
C GC C GAUGC C CUGGGCAGCAUGCUGAUCAGCUGGUACAUGAGC G GCUAC CACACA
GGAUACUACAUGGGCUUCAGACAGAACCAGAAGGAGGGCAGAUGCUCCCACUCCC
UGAACUGAY (SEQ ID NO: 4) 5' and 3' UTR Sequences X (5' UTR Sequence) =
GGACAGAUC GC CUGGAGAC GC CAUC CAC GCUGUUUUGAC CUC CAUAGAAGACAC C
GGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCG
UGCCAAGAGUGACUCACCGUCCUUGACACG (SEQ ID NO.: 5) Y (3' UTR Sequence) =
C GGGUGGCAUC C CUGUGAC C C CUC C C CAGUGC CUCUC CUG GC C CUGGAAGUUG C C
ACUC CAGUGC C CAC CAGC CUUGUC CUAAUAAAAUUAAGUUGCAUCAAGCU (SEQ
ID NO.: 6) Or GGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCA
CUC CAGUGC C CAC CAGC CUUGUC CUAAUAAAAUUAAGUUGCAUCAAAGCU (SEQ
ID NO.: 7) C-terminal His 10 Codon-Optimized Human CFTR mRNA ("His 10" disclosed as SEQ
ID NO: 11):
XAUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCA
UGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGAC
AUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAAC
GGGAAUG GGAC C G C GAACUC GC GUCUAAGAAAAACCCGAAGCUCAUCAACGCACU
GAGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGG
GGAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUAC
GACCCC GAUAACAAAGAAGAAC GGAGCAUC GC GAUCUAC CUC GGGAUCGGACUGU
GUUUGCUUUUCAUC GUCAGAACACUUUUGUUGCAUCCAGCAAUCUUC GGC CUC CA
UCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGAC
ACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUC
CCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUC
GUGUGGAUUGCCCCGUUGCAAGUC GC C CUUUUGAUGGGC CUUAUUUGGGAGCUG
UUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUU
CAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUC GC GAC CAGAGAGC GGGUAAA
AUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCA
AAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCC GC CAAA
CUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGC
GUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUG
AUUAAG GGGAUUAUC CUC C GCAAGAUUUUCAC CAC GAUUUC GUUCUGCAUUGUA
UUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGAC
211 UCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAG
ACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCU
UUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAAC
AACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGC
UCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUC
UCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUA
UGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAU
UCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUU
UCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGU
UGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAG
GGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGG
UAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACG
UAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUA
AGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGA
UCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCA
AAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAG
UUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGC
UUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGC
AGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACU
CAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUG
AAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGA
GCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUU
CAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGG
GGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACC
CCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACC
GGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUU
GAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUAC
AUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUC
UCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUU
GCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUC
ACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUC
UGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGU
CUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCC
ACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUU
GCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGC
UGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUG
UCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCA
GACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUAC
GCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCA
GCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGG
UUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUU
GUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGC
GAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUG
CAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCG
AGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACG
AAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCAC
212 GUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUG
ACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCA
UUAGC CC CGGUCAGCGUGUGGGGUUGCUCGGGAGGACC GGGUCAGGAAAAUCGA
CGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCG
ACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAG
UAAUC CC CCAAAAGGUCUUUAUCUUUAGCGGAAC CUUC CGAAAGAAUCUCGAUCC
UUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUC GCGGAC GAGGUUGGC CU
UCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGG
GGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUC
CUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGG
UAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGG
UGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUG
UCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGA
GAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCC
ACACAGAAAUUC GUCGAAGUGCAAGUCCAAACC GCAGAUC GC GGCCUUGAAAGAA
GAGACUGAAGAAGAAGUUCAAGACACGCGUCUUCACCAUCACCAUCACCAUCACC
AUCACCAUUAAY (SEQ ID NO.: 8) Codon-Optimized Human CFTR mRNA:
XAUGCAGCGGUC CC CGCUCGAAAAGGC CAGUGUCGUGUCCAAACUCUUCUUCUCA
UGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGAC
AUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAAC
GGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACU
GAGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGG
GGAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUAC
GACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGU
GUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCA
UCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGAC
ACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUC
CCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUC
GUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUG
UUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUU
CAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAA
AUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCA
AAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAA
CUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGC
GUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUG
AUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUA
UUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGAC
UCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAG
ACC CUGGAGUACAAUCUUACUACUAC GGAGGUAGUAAUGGAGAAUGUGACGGCU
UUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAAC
AACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGC
UCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUC
UCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUA
UGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAU
213 UCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUU
UCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGU
UGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAG
GGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGG
UAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACG
UAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUA
AGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGA
UCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCA
AAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAG
UUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGC
UUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGC
AGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACU
CAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUG
AAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGA
GCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUU
CAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGG
GGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACC
CCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACC
GGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUU
GAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUAC
AUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUC
UCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUU
GCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUC
ACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUC
UGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGU
CUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCC
ACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUU
GCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGC
UGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUG
UCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCA
GACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUAC
GCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCA
GCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGG
UUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUU
GUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGC
GAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUG
CAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCG
AGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACG
AAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCAC
GUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUG
ACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCA
UUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGA
CGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCG
ACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAG
UAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCC
UUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCU
214 UCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGG
GGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUC
CUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGG
UAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGG
UGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUG
UCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGA
GAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCC
ACACAGAAAUUC GUCGAAGUGCAAGUCCAAACC GCAGAUC GC GGCCUUGAAAGAA
GAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAAY (SEQ ID NO.: 9) Codon Optimized Human CFTR mRNA coding sequence with a Growth Hormone Leader Sequence (italisized and underlined):
AUGGCCACUGGAUCAAGAACCUCACUGCUGCUCGCUUUUGGACUGCUUUGCCUGCC
CUGGUUGCAAGAAGGAUCGGCUUUCCCGACCAUCCCACUCUCCAUGCAGCGGUCCC
CGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACUCGGCCUAU
CCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAUCCCC
UCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGC
GAACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCU
UCUGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAG
CAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAA
AGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAU
CGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAUG
CAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUCUCG
UCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAU
AACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCC
CGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGUUGCAGGCAUCUG
CCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUG
GGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGAC
UCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUG
GGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACU
GACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCC
GGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUU
AUC CUCC GCAAGAUUUUCAC CAC GAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAG
UGACACGGCAAUUUC CGUGGGC CGUGCAGACAUGGUAUGACUCGCUUGGAGC GA
UCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUACA
AUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGG
GUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCU
CAAAUGGGGACGAUUCC CUGUUUUUCUC GAACUUCUCC CUGCUCGGAACACCC GU
GUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGG
AAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGA
GCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUU
UCAUGGAUCAUGC CC GGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAU
GAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUU
UCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUG
UCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCA
215 GAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAAAA
GAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUG
GUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACG
AAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGCAGCCGGA
CUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCAGCGCGGAACGG
CGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCC
CGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUUG
GUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCU
CAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACG
AACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAU
UCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGG
CAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACC
GCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGAAUUUGAC
UGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGGACUUGAGAUCAGC
GAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUCA
AUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGGUGCACAAGU
CCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGCUGAGGUCGCAGC
GUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAAAGGCAAU
UCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGU
AUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUU
UCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCA
CCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUC
AAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGAC
CUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGG
CUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUGUCGCGACCGUUCCGGU
GAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCUU
AAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUACGCAUCUUGUGACCAGU
UUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACUUUGAAACA
CUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGU
ACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUC
GCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGU
AUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAAC
AGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUC
AUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAU
GGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGAC
AUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCG
AGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCG
UGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUU
CUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGA
UAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUC
UUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCA
GAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAG
CAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCG
CAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAA
UUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAU
CAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCA
216 UCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAA
GGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGG
CAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCGUCGA
AGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAG
UUCAAGACACGCGUCUUUAA (SEQ ID NO: 10) [0430] Aliquots of 50 mg/mL ethanolic solutions of cKK-E12, DOPE, Chol and DMG-PEG2K were mixed in a molar ratio of 40:30:25:5 and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaC1, pH 4.5) of FIX, ASS1, or FFL mRNA was prepared from a 1 mg/mL stock. The lipid solution was injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol.
The resulting nanoparticle suspension was filtered, diafiltrated with lx PBS
(pH 7.4), concentrated and stored at 2-8 C. The final concentration of FIX mRNA was approximately 0.77 mg/mL FIX mRNA (encapsulated), Zave = 76 nm, PDI = 0.08. The final concentration of ASS1 mRNA was approximately 0.64 mg/mL ASS1 mRNA (encapsulated), Zave = 78 nm (Dv(50) = 46 nm; Dv(90) = 96 nm). The final concentration of FFL mRNA was approximately 1.31 mg/mL FFL mRNA (encapsulated), Zave = 75 nm, PDI ¨ 0.11. The final concentration of SMN mRNA was approximately 1.85 mg/mL SMN mRNA (encapsulated). Average particle size (Zave) = 71 nm, (particle size for 50% of particles was 44nm or less (Dv(50)) = 44 nm; and the particle size for 90% of the particles was 93n or less (Dv(90) = 93 nm)).
Example 2. Administration of mRNA-loaded Liposome Nanoparticles [0431] This example illustrates exemplary methods of administering mRNA-loaded liposome nanoparticles and methods for analyzing delivered mRNA and subsequently expressed protein in various target tissues in vivo.
[0432] All studies were performed using male CD-1 mice of approximately 6-8 weeks of age at the beginning of each experiment. Samples were introduced by a single bolus tail-vein injection of an equivalent total dose of 1.0 mg/kg (or otherwise specified) of encapsulated FIX, FFL or ASS1 mRNA. Mice were sacrificed and perfused with saline at the designated time points.
217 [0433] Various organ tissues such as the liver, spleen, kidney and heart of each mouse was harvested, apportioned into separate parts, and stored in either 10%
neutral buffered formalin or snap-frozen and stored at -80 C for analysis.
[0434] All animals were euthanized by CO2 asphyxiation at designated time points post dose administration ( 5%) followed by thoracotomy and terminal cardiac blood collection.
Whole blood (maximal obtainable volume) was collected via cardiac puncture on euthanized animals into serum separator tubes, allowed to clot at room temperature for at least 30 minutes, centrifuged at 22 C 5 C at 9300 g for 10 minutes, and the serum extracted.
For interim blood collections, approximately 40-504 of whole blood was collected via facial vein puncture or tail snip. Samples collected from non-treatment animals were used as a baseline ASS1 levels for comparison to study animals.
Enzyme-Linked Immunosorbent Assay (ELISA) Analysis A. Human FIX ELISA
[0435] Quantification of FIX protein was performed following procedures reported for human FIX ELISA kit (AssayMax, Assay Pro, Catalog # EF1009-1).
B. Human ASS] ELISA
[0436] Standard ELISA procedures were followed employing mouse anti-ASS1 2E12 IgG as the capture antibody with rabbit anti-ASS1 #3285 IgG as the secondary (detection) antibody (Shire Human Genetic Therapies). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was used for activation of the 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution. The reaction was quenched using 2N H2504 after 20 minutes. Detection was monitored via absorption (450 nm) on a Molecular Device SpectraMax instrument.
Untreated mouse serum and organs and human ASS1 protein were used as negative and positive controls, respectively.
IVIS Bioluminometer Measurements
218 [0437] To visual luminescence in treated mice, several steps were followed. Anesthesia using isoflurane vaporizer at 1 - 3 % (usually @2.5%) was initially employed.
Using a microsprayer, 50 4/animal of luciferin in PBS was administered at 60 mg/mL via intratracheal/intranasal. Luciferin was allowed to distribute for 5-10 minutes. Animals were placed in an isoflurane chamber until anesthetized. Anesthetized animals were placed into the IVIS imaging chamber at dorsal recumbency and positioned into the manifold.
Pictures of mice were taken. In these Examples, the acquisition settings providing highest sensitivity were:
camera height at D level, F/Stop at fl, binning at high resolution, and exposure time at 5 minutes. Exposures were repeated up to 3 times (5, 10 and 15 minutes post Luciferin Injection).
In Situ Hybridization (ISH) Analysis [0438] In situ hybridization was performed using "ZZ" probe technology.
Probes were generated based on codon-optimized sequence of human messenger RNA. Tissues were fixed for 24-48 hours in 10% neutral buffered formalin and embedded in paraffin.
Positive detection of desired mRNA was achieved through 6 consecutive amplification steps followed by chromagenic visualization using 3,3'-diaminobenzidine (DAB). Positive signal was compared to that of untreated mouse.
Example 3. Highly Effective in vivo Production of Therapeutic Proteins [0439] This example demonstrates highly efficient and sustained production of proteins encoded by mRNA delivered by liposomes incorporating the cationic lipids described herein (e.g., cKK-E12) in serum and various organ tissues.
In Vivo Human FIX Protein Production Results [0440] The production of human FIX protein via hFIX mRNA-loaded cKK-E12-based lipid nanoparticles was tested in CD-1 mice as a single, bolus intravenous injection. Figure 1 represents the amount of human FIX protein detected via ELISA when treating mice with human FIX mRNA-loaded cKK-E12-based lipid nanoparticles as compared to a C12-200-based lipid nanoparticle encapsulating hFIX mRNA. The mice were sacrificed twenty-four hours post-injection and organs were harvested (as described above).
219 [0441] C12-200-based lipid nanoparticles have been shown to be an effective vehicle to deliver and express mRNA in vivo (see, PCT Application Publication NO.
W02012170930, the disclosure of which is hereby incorporated by reference). Surprisingly, as represented in Figure 1, cKK-E12 based lipid nanoparticles are even more effective in delivering human FIX mRNA
in vivo, resulting in close to 50% higher protein expression detected in the plasma of the treated mice, as compared to C12-200-based lipid nanoparticles.
[0442] Figure 2 shows the results of a dose-response experiment as represented by the amount of human FIX protein detected via ELISA when treating mice with human FIX mRNA-loaded cKK-E12-based lipid nanoparticles at various doses. The mice were bled at 6 hours and sacrificed twenty-four hours post-injection and organs were harvested (as described above).
[0443] A clear dose response was achieved when measuring liver levels of human FIX
protein. The dosing range was from 0.10 ¨ 3.0 mg/kg of encapsulated human FIX
mRNA.
These data demonstrate the ability of the lipid nanoparticles to efficiently deliver messenger RNA, release the payload and process this exogenous mRNA via translation to produce human FIX protein, which is then subsequently secreted into the bloodstream. Levels of human FIX
protein are well above therapeutic levels (>100 ng/mL plasma) and surpass normal physiological levels (-5 ug/mL plasma) when dosing at 1.0 mg/kg or greater. Further, the plasma residence time of this human protein is sustained through at least 24 hours post administration.
In Vivo Human ASS] Protein Production Results [0444] The production of human ASS1 protein via codon-optimized hASS1 mRNA-loaded cKK-E12-based lipid nanoparticles was tested in CD-1 mice as a single, bolus intravenous injection. Figure 3 represents the amount of human ASS1 protein detected via ELISA when treating mice with human ASS1 mRNA-loaded cKK-E12-based lipid nanoparticles at various doses. The mice were sacrificed twenty-four hours post-injection and organs were harvested (as described above).
[0445] A clear dose response was achieved when measuring liver levels of human ASS1 protein. As shown in Table 5, the dosing range was from 0.10 ¨2.0 mg/kg of encapsulated human ASS1 mRNA in cKK-E12 lipid nanoparticles. These data demonstrate the ability of the
220 lipid nanoparticles to accumulate in the liver and release the mRNA payload and the liver to process this exogenous mRNA via translation to produce human ASS1 protein.
Dose Encapsulated Human ASS! mRNA (mg/kg) ASS! Protein (ng/mg total protein) 0.10 BLD
0.30 BLD
0.60 546 1.0 1388 2.0 3371 Table 5. Raw values of human ASS1 protein as measured via ELISA analysis (as depicted in Figure 1). Codon-optimized human ASS1 mRNA was delivered via cKK-E12-based lipid nanoparticles. Doses are based on encapsulated ASS1 mRNA. Values are depicted as nanogram of human ASS1 protein per milligram total protein in liver. BLD = Below Limit of Detection for ELISA.
[0446] While the sensitivity of the ELISA has limitations at lower values, western blot analysis allows for clear visualization of the human ASS1 protein at lower doses (0.3 ¨ 3.0 mg/kg) (see Figure 4). Figure 4 depicts a comparison of human ASS1 protein levels in liver as a function of dose via western blot analysis upon a single intravenous dose of human ASS1 mRNA-encapsulated cKK-E12 lipid nanoparticles. CD1 mice were sacrificed at 24 hours post-administration and livers were harvested and analyzed as described above.
[0447] To further understand the ability of ASS1 mRNA-encapsulated lipid nanoparticles to facilitate the delivery of mRNA to selected organs (liver), a pharmacokinetic analysis was performed, monitoring human ASS1 protein levels in the liver over a one week time period.
Figure 5 depicts the quantity of human ASS1 protein detected in the liver at various time points up to 7 days after administration of human ASS1-loaded lipid nanoparticles (cKK-E12). This was accomplished as a single dose (1.0 mg/kg encapsulated mRNA) given intravenously.
[0448] In this case, we observed a maximum serum level of human ASS1 protein at approximately 24-48 hours post-administration. Measurable levels of protein were still observed 1 week post-administration as determined by both ELISA and western blot (Figures 5 and 6, respectively). Figure 6 depicts a comparison of human ASS1 protein levels in liver over time
221 via western blot analysis upon a single intravenous dose of human ASS1 mRNA-encapsulated lipid nanoparticles (1.0 mg/kg dose).
[0449] Direct detection of the active pharmaceutical ingredient (ASS1 mRNA) in the livers of the treated mice was achieved using in situ hybridization (ISH) based methods. As demonstrated in Figures 7 & 8, the exogenous human ASS1 messenger RNA could be detected in high levels at the earliest time point tested (30 minutes) and the signal remained strong for 48 hours after dosing. Further, human ASS1 mRNA was still detectable 72 hours post-administration.
[0450] In addition to ISH, detection of the resulting human AS 51 protein was achieved using immunohistochemical (IHC) means. Using a mouse monoclonal antibody (02D2-2E12) for specific binding, the presence of target human ASS1 protein in the cytoplasm of hepatocytes of treated livers can be readily observed. Figure 9 shows the immunohistochemical staining of human ASS1 protein in treated mouse livers 24 hours after administration.
In vivo delivery of EEL mRNA via nebulization [0451] To assess whether additional routes of delivery were feasible, FFL
mRNA was encapsulated in cKK-E12 liposomes and those liposomes were nebulized. As shown in Figure 10, it is possible to efficiently nebulize cKK-E12 based lipid nanoparticles encapsulating mRNA.
Figure 10 represents mice treated with luciferin 24 hours after exposure to nebulized FFL
mRNA loaded cKK-E12 lipid nanoparticles.
Example 4. CNS Delivery of hSIVIN-1 mRNA
[0452] This example provides an exemplary cKK-E12 liposome formulations for effective delivery and expression of mRNA in the CNS. Specifically, the example demonstrates that delivery of human survival of motor neuron-1 (hSMN-1) mRNA into various tissues of the brain and spinal cord.
Messenger RNA Material
222 [0453] Codon-optimized human Survival of Motor Neuron-1(hSMN-1) messenger RNA
(see SEQ ID NO: 4) was synthesized by in vitro transcription from a plasmid DNA template encoding the gene, which was followed by the addition of a 5' cap structure (Cap 1) (Fechter, P.;
Brownlee, G.G. "Recognition of mRNA cap structures by viral and cellular proteins" J. Gen.
Virology 2005, 86, 1239-1249) and a 3' poly(A) tail of approximately 250 nucleotides in length (SEQ ID NO: 15) as determined by gel electrophoresis. The 5' and 3' untranslated regions present in each mRNA product are represented as X and Y, respectively and defined as stated in Example 1.
Formulation Protocol [0454] Lipid nanoparticles (LNP) were formed via standard ethanol injection methods (Ponsa, M.; Foradada, M.; Estelrich, J. "Liposomes obtained by the ethanol injection method"
Int. J. Pharm. 1993, 95, 51-56). For the various lipid components, a 50mg/m1 ethanolic stock solutions was prepared and stored at -20 C. In preparation of the cKK-E12 lipid nanoparticle formulation listed in Table 6, each indicated lipid component was added to an ethanol solution to achieve a predetermined final concentration and molar ratio, and scaled to a 3 ml final volume of ethanol. Separately, an aqueous buffered solution (10mM citrate/150 mM NaC1, pH 4.5) of hSMN-1 mRNA was prepared from a 1 mg/ml stock. The lipid solution was injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20%
ethanol. The resulting nanoparticle suspension was filtered and dialysed against 1xPBS (pH
7.4), concentrated and stored between 2-8 C. SMN-1 mRNA concentration was determined via the Ribogreen assay (Invitrogen). Encapsulation of mRNA was calculated by performing the Ribogreen assay with and without the presence of 0.1% Triton-X 100. Particle sizes (dynamic light scattering (DLS)) and zeta potentials were determined using a Malvern Zetasizer instrument in lx PBS and 1mM KC1 solutions, respectively.
Table 6. Exemplary cKK-E12 Lipid Nanoparticle formulation Formulations Components Molar Ratio Final mRNA
Zeta Parameters of lipids Concentration
223 cKK-E12 DOPE Zaõ = 72 nm;
1 Cholesterol 40:30:25:5 1.8 mg/ml Dv(50) = 49 nm;
DMG-PEG-2K Dv(90) =90 nm hSMN-1 mRNA
Intrathecal administration of mRNA loaded liposome nanoparticles [0455] All in vivo studies were performed using either rats or mice of approximately 6-8 weeks of age at the beginning of each experiment. At the start of the experiment, each animal was anesthetized with isoflurane (1-3%, to effect) by inhalation. Once anesthetized, each animal was shaved at the exact injection site (L4-L5 or L5-L6). Following insertion of the needle, reflexive flick of the tail was used to indicate puncture of the dura and confirm intrathecal placement. Each animal received a single bolus intrathecal injection of the test formulation listed in Table 6. All animals were sacrificed 24 hours post injection and perfused with saline.
Isolation of organ tissues for analysis [0456] All animals had the whole brain and spinal cord harvested. The brain was cut longitudinally and placed in one histology cassette per animal. The whole spinal cord was stored ambient in a 15 ml tube containing 10% neutral buffered formalin (NBF) for at least 24 hours and no more than 72 hours before transfer into 70% histology grade alcohol solution. Each spinal cord sample was cut into cervical, thoracic and lumbar sections. Each spinal cord section cut in half and both halves were placed in individual cassettes per section (cervical, thoracic and lumbar) for processing. All three cassettes were embedded into one paraffin block per animal.
When applicable, portions of brain and spinal cord were snap frozen and stored at -80 C.
hSMN-1 Western Blot Analysis [0457] Standard western blot procedures were followed employing various antibodies that recognizes hSMN protein, such as: (A) anti-SMN 4F11 antibody at 1:1,000 dilution; (B)
224 Pierce PA5-27309 a-SMN antibody at 1:1,000 dilution; and (C) LSBio C138149 a-SMN
antibody at 1:1,000 dilution. For each experiment one microgram of hSMN mRNA
was transfected into ¨1x106 BHK-21 cells using Lipofectamine 2000. Cells were treated with OptiMem and harvested 16-18 hours post-transfection. Cell lysates were harvested, processed and loaded on to an 8-16% Tris Glycine gel. The gel was transferred using a PVDF membrane and treated with the respective primary antibody. Goat anti-mouse HRP antibody was used as the secondary antibody at 1:10,000 dilution for 45 minutes at room temperature followed by washing and development. The data demonstrates that each antibody tested showed a strong signal for hSMN-1 and was specific for human SMN, as indicated by an absence in a cross-reactive signal for untreated BHK cells (Figure 11).
In Situ Hybridzation (ISH) Analysis [0458] Tissue from each representative sample, was assayed for hSMN-1 mRNA using a manual in situ hybridization analysis, performed using RNAscope0 (Advanced Cell Diagnostic) "ZZ" probe technology. Probes were generated based on the codon-optimized sequence of human SMN messenger RNA (SEQ ID NO: 4). Briefly, the RNAscope0 assay is an in situ hybridication assay designed to visualize single RNA molecules per cell in formalin-fixed, paraffin-embedded (FFPE) tissue mounted on slides. Each embedded tissue sample was pretreated according to the manufacturers protocol and incubated with a target specific hSMN-1 RNA probe. The hSMN-1 probe was shown to be specific for human SMN-1 and had little to no cross reactivity with mouse or rat SMN-1. Once bound, the hSMN-1 probe is hybridized to a cascade of signal amplification molecules, through a series of 6 consecutive rounds of amplification. The sample was then treated with an HRP-labeled probe specific to the signal amplification cassette and assayed by chromatic visualization using 3,3'-diaminobenzidine (DAB). A probe specific for Ubiquitin C was used as the positive control.
Positive SMN signal was compared to that of untreated and vehicle control treated rat or mouse tissue. Stained samples were visualized under a standard bright field microscope.
Immunohistochemical Analysis
225 [0459] Human SMN-1 mRNA-loaded lipid nanoparticles were administered to rats via intrathecal injection, and tissue samples collected and processed 24 hours post administration in accordance with the methods described above. Rat spinal tissue samples were then assayed for hSMN-1 protein expression. Briefly, fixed tissue embedded in paraffin was processed and placed on slides. The slides were dewaxed, rehydrated and antigen retrieval was performed using a pressure cooker with citrate buffer. Several blocking buffers were employed followed by primary antibody incubation overnight at 4 C, using the 4F11 antibody at a 1:2500 dilution. The resulting slides were washed and incubated at ambient temperature with the secondary antibody polymer followed by washing and subsequent chromagen development. The data demonstrates that in as little as 24 hours post intrathecal adminiatration of hSMN-1 mRNA, staining is observed for human SMN-1 protein when compared to no-treatment control (Figure 13). This supports the previous findings which demonstrate delivery of hSMN-1 mRNA to the spinal tissue. Furthermore, the data demonstrates that once delivered to the cell hSMN-1 mRNA is effectively expressed to generate hSMN-1 protein.
Results [0460] The data presented in this example demonstrates that intrathecal administration of hSMN-1 mRNA loaded liposomes (e.g., lipid or polymer-based nanoparticles) results in successful intracellular delivery of mRNA in neurons in the brain and spinal cord, including those difficult to treat cells, such as anterior horn cells and dorsal root ganglia.
[0461] The results have shown that mRNA encapsulated within a lipid nanoparticle (e.g., lipid nanoparticle comprising cKK-E12) can be effectively delivered to various tissues of the CNS following intrathecal administrations. Using the exemplary formulation disclosed in Table 6, mRNA was effectively delivered and internalized within various neurons of the spinal cord (Figures 12A-12C), as verified by in situ hybridization assay. Surprisingly, intracellular mRNA
delivery was demonstrated in the difficult to reach neuronal cells of the anterior horn, located deep within the tissues of the spinal column (Figures 12A-12C). Little to no background was observed with mouse or rat SMN-1, indicating specificity for the human SMN-1 probe. Positive
226 SMN signal was compared to that of untreated and vehicle control treated rat or mouse tissue.
Stained samples were visualized under a standard bright field microscope.
[0462] These data demonstrates that the lipid or polymer nanoparticle based mRNA
delivery approach described herein was able to successfully permeate the complex and dense cell membrane of the spinal cord neurons and deliver the mRNA payload for the production of encoded proteins inside neurons. It was particularly surprising that the mRNA
delivery approach described herein was equally successful in permeating difficult to treat neurons such as anterior horn cell and dorsal root ganglia. Thus, the data presented herein demonstrates that lipid or polymer nanoparticles, such as those comprising cKK-E12, may serve as a promising option for delivering mRNA to neuronal cells in the treatment of a CNS disease. In particular, the present example demonstrates that hSMN mRNA loaded nanoparticles can be effectively delivered to neurons, including those difficult to treat motor neurons in the spinal cord, and can be used for the production of SMN protein and treatment of spinal muscular atrophy.
Example 5. In Vivo CO-CFTR-C-Hisio mRNA Delivery to CFTR Knockout Mice [0463] Messenger RNA Synthesis. For the experiment, C-terminal Hisio tagged codon-optimized human cystic fibrosis transmembrane conductance regulator (CO-CFTR-C-Hisio) (SEQ ID NO:8) ("Hisio" disclosed as SEQ ID NO: 11) and non-tagged codon-optimized human CFTR (CO-CFTR) (SEQ ID NO:9) mRNA were synthesized by in vitro transcription from a plasmid DNA template using standard method. mRNAs used in this example and Example 6 were produced by IVT in which 25% of U residues were 2-thio-uridine and 25% of C residues were 5-methylcytidine.
[0464] Analysis of human CFTR protein produced via intratracheal administered mRNA-loaded nanoparticles. For the study, CFTR knockout mice were used. CFTR mRNA
formulation or vehicle control was introduced using a PARI Boy jet nebulizer.
Mice were sacrificed and perfused with saline, after a predetermined period of time, to allow for protein expression from the mRNA.
[0465] PEI Formulation. PEI formulation has been used to deliver CFTR
mRNA to the lung and was used as a control in this experiment. Polymeric nanoparticle formulations with 25
227 kDa branched PEI were prepared as follows. The required amount of mRNA was diluted just before application in water for injection (Braun, Melsungen) to a total volume of 4 ml and added quickly to 4 ml of an aqueous solution of branched PEI 25 kDa using a pipette at an N/P ratio of 10. The solution was mixed by pipetting up and down ten times and nebulized as two separate 4.0 ml fractions one after another to the mouse lungs using the indicated nebulizer.
[0466] cKK-E12 Formulation. For the lipid-based nanoparticle experiment, a lipid formulation was created using CO-CFTR-C-Hisio RNA in a formulation of cKK-E12:DOPE:Chol:PEGDMG2K (relative amounts 50:25:20:5 (mg:mg:mg:mg)). The solution was nebulized to the mouse lungs using the indicated nebulizer.
[0467] Nebulization (Aerosol) Administration of Human CO-CFTR-C-His 10 mRNA.
CFTR test materials were administered by a single aerosol inhalation via PARI
Boy jet nebulizer (nominal dose volume of up to 8 mL/group). The test material was delivered to a box containing the whole group of animals (n=4) and connected to oxygen flow and scavenger system.
[0468] Administration of Human CO-CFTR-C-His 10 mRNA. CFTR mRNA was prepared in the manner described above. Four CFTR knockout mice were placed in an aerosol chamber box and exposed to 2 mg total codon optimized unmodified human CFTR mRNA
(comprising the coding sequence of SEQ ID NO: 8) via nebulization (Pan i Boy jet nebulizer) over the course of approximately one hour. Mice were sacrificed 24 hours post-exposure.
[0469] Euthanasia. Animals were euthanized by CO2 asphyxiation at representative times post-dose administration ( 5%) followed by thoracotomy and exsanguinations. Whole blood (maximal obtainable volume) was collected via cardiac puncture and discarded.
[0470] Perfusion. Following exsanguination, all animals underwent cardiac perfusion with saline. In brief, whole body intracardiac perfusion was performed by inserting 23/21 gauge needle attached to 10 mL syringe containing saline set into the lumen of the left ventricle for perfusion. The right atrium was incised to provide a drainage outlet for perfusate. Gentle and steady pressure was applied to the plunger to perfuse the animal after the needle had been positioned in the heart. Adequate flow of the flushing solution was ensured when the exiting perfusate flows clear (free of visible blood) indicating that the flushing solution has saturated the body and the procedure was complete.
228 [0471] Tissue Collection. Following perfusion, all animals had their lungs (right and left) harvested. Both (right and left) lungs were snap frozen in liquid nitrogen and stored separately at nominally -70 C.
[0472] Expression of human CFTR in CO-CFTR-C-His 10 in CFTR knockout mice.
CFTR expression was detected by Western blot analysis of tissue lysate collected from CFTR
mRNA-treated mouse lungs. Mature "C" band was detected in left and right lungs of all treated mice, for both the cKK-E12-based and PEI-based formulations (Figure 14).
Expression of the mature "C" band was verified by comparison with lysate collected from HEK 293T
human CO-CFTR-C-Hisio positive cells. In contrast, no detectable signal was observed in lysate collected from wild type untreated control mice (Figure 14). Taken together, these data suggest that cKK-E12 may be used to deliver mRNA (e.g., CFTR mRNA) to the lung via, e.g., inhalation, as effectively as or even better than PEI based formulations.
Example 6: In Vivo Expression in the Lung [0473] This example further desmonstrates successful in vivo expression in the lung following aerosol delivery of mRNA-loaded ckk-E12 based nanoparticles. All studies were performed using pigs of the German Landrace, obtained from Technical University Munich, Weihenstephan, Germany. The pigs had a body weight ranging from 35-90 kg.
FFL/CO-CFTR-C-His10 mRNA formulation or vehicle control was introduced using a Pan i jet nebulizer. Pigs were sacrificed and perfused with saline, after a predetermined period of time, to allow for protein expression from the mRNA.
[0474] Messenger RNA Synthesis. In the example, codon optimized fire fly luciferase (CO-FFL) mRNA was synthesized by in vitro transcription from plasmid DNA
templates.
[0475] cKK-E12 Formulation. For the lipid-based nanoparticle experiment, a lipid formulation was created using 1 mg FFL + 9 mg of CO-CFTR-C-Hisio mRNA
encapsulated in a formulation of cKK-E12:DOPE:Chol:PEGDMG2K (relative amounts 40:30:25:5 (mol ratio).
The solution was nebulized to the Pig lungs using the indicated nebulizer.
229 [0476] Aerosol Application. The aerosol (Saline or CO-FFL cKK-E12 formulation) was nebulized and inhaled into the anaesthetized pig. Sedation in pigs was initiated by premedication with azaperone 2 mg/kg body weight, ketamine 15 mg/kg body weight, atropine 0.1 mg/kg body weight and followed by insertion of an intravenous line to the lateral auricular vein. Pigs were anesthetized by intravenous injection of propofol 3-5 mg/kg body weight as required.
Anesthesia was maintained by isoflurane (2-3%) with 1% propofol bolus injection at 4 to 8 mg/kg body weight to enhance anesthesia as required. Duration of the anesthesia was approximately 1-3 hrs. Pigs were killed with bolus injection of pentobarbital (100 mg/kg body weight) and potassium chloride via the lateral ear vein. Lungs were excised and tissue specimens were collected from various lung regions followed by incubation in cell culture medium overnight. The stored samples were subjected to bioluminescence detection.
[0477] Bioluminescence Analysis. For measurement of luciferase activity, tissue specimens were either homogenized and analyzed in a tube luminometer or incubated in a medium bath comprising D-Luciferin substrate and subjected to ex vivo luciferase BLI. The data illustrate that a strong bioluminescence signal was observed for each of the (A) CO-FFL/
CO-CFTR-C-Hisio mRNA treated pigs, when compared to (B) control lung tissue samples from control pigs (Saline vehicle control) (Figure 15 A&B).
[0478] These data illustrate that FFL/CFTR mRNA were successfully delivered to and expressed in the lung by aerosol administration of a cKK-E12 based lipid formulation.
230 EQUIVALENTS
[0479] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:
231

Claims (56)

We claim:
1. A method of delivery of messenger RNA (mRNA) in vivo, comprising administering to a subject in need of delivery a composition comprising an mRNA encoding a protein, encapsulated within a liposome such that the administering of the composition results in the expression of the protein encoded by the mRNA in vivo;
wherein the liposome comprises a cationic lipid of formula I-c:
or a pharmaceutically acceptable salt thereof, wherein:
p is an integer of between 1 and 9, inclusive;
each instance of R2 is independently hydrogen or optionally substituted C1-6 alkyl;
each instance of R6 and R7 is independently a group of the formula (i), (ii), or (iii);
Formulae (i), (ii), and (iii) are:
(i) (ii) (iii) wherein:
each instance of R' is independently hydrogen or optionally substituted alkyl;
X is O, S, or NR X, wherein R X is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;
Y is O, S, or NR Y, wherein R Y is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;
R P is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom; and R L is optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted heteroC1-50 alkyl, optionally substituted heteroC2-50 alkenyl, optionally substituted heteroC2-50 alkynyl, or a polymer.
2. The method of claim 1, wherein the cationic lipid is cKK-E12:

3. The method of any one of the preceding claims, wherein the liposome further comprises one or more non-cationic lipids, one or more cholesterol-based lipids and/or one or more PEG-modified lipids.
4. The method of claim 3, wherein the one or more non-cationic lipids are selected from DSPC
(1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC
(1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)).
5. The method of claim 3 or 4, wherein the one or more cholesterol-based lipids are cholesterol and/or PEGylated cholesterol.
6. The method of any one of claims 3-5, wherein the one or more PEG-modified lipids comprise a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length.
7. The method of any one of the preceding claims, wherein the liposome comprises cKK-E12, DOPE, cholesterol and DMG-PEG2K.
8. The method of any one of the preceding claims, wherein the cationic lipid constitutes about 30-50 % of the liposome by molar ratio.
9. The method of claim 8, wherein the cationic lipid constitutes about 40 % of the liposome by molar ratio.
10. The method of any one of claims 7-9, wherein the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:30:20:10 by molar ratio.
11. The method of any one of claims 7-9, wherein the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:30:25:5 by molar ratio.
12. The method of any one of claims 7-9, wherein the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:32:25:3 by molar ratio.
13. The method of any one of the preceding claims, wherein the liposome has a size less than about 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, or 50 nm.
14. The method of any one of the preceding claims, wherein the composition is administered intravenously.
15. The method of any one of claims 1-13, wherein the composition is administered via pulmonary delivery.
16. The method of claim 15, wherein the pulmonary delivery is by aerosolization, inhalation, nebulization or instillation.
17. The method of any one of claims 1-13, wherein the composition is administered intrathecally.
18. The method of any one of the preceding claims, wherein the expression of the protein encoded by the mRNA is detectable in liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and cerebrospinal fluid.
19. The method of any one of the preceding claims, wherein the expression of the protein encoded by the mRNA is detectable 3 hours after the administration.
20. The method of any one of the preceding claims, wherein the expression of the protein encoded by the mRNA is detectable 6 hours after the administration.
21. The method of any one of the preceding claims, wherein the expression of the protein encoded by the mRNA is detectable 12 hours after the administration.
22. The method of any one of the preceding claims, wherein the expression of the protein encoded by the mRNA is detectable 24 hours after the administration.
23. The method of any one of the preceding claims, wherein the expression of the protein encoded by the mRNA is detectable 1 week after the administration.
24. The method of any one of the preceding claims, wherein the mRNA has a length of or greater than about 0.5kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb.
25. The method of any one of the preceding claims, wherein the protein encoded by the mRNA
is a cytosolic protein.
26. The method of any one of claims 1-24, wherein the protein encoded by the mRNA is a secreted protein.
27. The method of any one of the preceding claims, wherein the protein encoded by the mRNA
is an enzyme.
28. The method of any one of the preceding claims, wherein the protein encoded by the mRNA
is Argininosuccinate Synthetase (ASS1), Factor IX, survival of motor neuron 1, or phenylalanine hydroxylase.
29. The method of any one of the preceding claims, wherein the mRNA is administered at a dose ranging from about 0.1 ¨ 2.0 mg /kg body weight.
30. The method of any one of claims 1-28, wherein the mRNA is administered at a dose of or less than about 1.0 mg /kg body weight.
31. The method of any one of claims 1-28, wherein the mRNA is administered at a dose of or less than about 0.5 mg /kg body weight.
32. The method of any one of claims 1-28, wherein the mRNA is administered at a dose of or less than about 0.3 mg/kg body weight.
33. The method of any one of the preceding claims, wherein the mRNA comprises one or more modified nucleotides.
34. The method of claim 33, wherein the one or more modified nucleotides comprise pseudouridine, N-1-methyl-pseudouridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and/or 2-thiocytidine.
35. The method of any one of claims 1-32, wherein the mRNA is unmodified.
36. A method of treating a disease or disorder comprising administering to subject in need of treatment a composition comprising an mRNA

encoding a therapeutic protein encapsulated within a liposome such that the administering of the composition results in the expression of the protein encoded by the mRNA in one or more tissues affected by the disease or disorder;
wherein the liposome comprises a cationic lipid of formula I-c:

or a pharmaceutically acceptable salt thereof, wherein:
p is an integer of between 1 and 9, inclusive;
each instance of R2 is independently hydrogen or optionally substituted C1-6 alkyl;
each instance of R6 and R7 is independently a group of the formula (i), (ii), or (iii);
Formulae (i), (ii), and (iii) are:
wherein:
each instance of R' is independently hydrogen or optionally substituted alkyl;
X is O, S, or NR X, wherein R X is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;
Y is O, S, or NR Y, wherein R Y is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;
R P is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom; and R L is optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted heteroC1-50 alkyl, optionally substituted heteroC2-50 alkenyl, optionally substituted heteroC2-50 alkynyl, or a polymer.
37. The method of claim 36, wherein the cationic lipid is cKK-E12:
38. A composition for delivery of messenger RNA (mRNA) comprising an mRNA
encoding a protein encapsulated within a liposome, wherein the liposome comprises a cationic lipid of formula I-c:

or a pharmaceutically acceptable salt thereof, wherein:
p is an integer of between 1 and 9, inclusive;
each instance of R2 is independently hydrogen or optionally substituted C1-6 alkyl;
each instance of R6 and R7 is independently a group of the formula (i), (ii), or (iii);
Formulae (i), (ii), and (iii) are:
wherein:
each instance of R' is independently hydrogen or optionally substituted alkyl;
X is O, S, or NR X, wherein R X is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;
Y is O, S, or NR Y, wherein R Y is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;
R P is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom; and R L is optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted heteroC1-50 alkyl, optionally substituted heteroC2-50 alkenyl, optionally substituted heteroC2-50 alkynyl, or a polymer.
39. The composition of claim 38, wherein the cationic lipid is cKK-E12:
40. The composition of claim 38 or 39, wherein the liposome further comprises one or more non-cationic lipids, one or more cholesterol-based lipids and/or one or more PEG-modified lipids.
41. The composition of any one of claims 38-40, wherein the one or more non-cationic lipids are selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC
(1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)).
42. The composition of claim 40 or 41, wherein the liposome further comprises one or more non-cationic lipids, one or more cholesterol-based lipids and/or one or more PEG-modified lipids.
43. The composition of any one of claims 40-42, wherein the one or more PEG-modified lipids comprise a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length.
44. The composition of any one of claims 40-43, wherein the liposome comprises cKK-E12, DOPE, cholesterol and DMG-PEG2K.
45. The composition of any one of claims 40-44, wherein the cationic lipid constitutes about 30-50 % of the liposome by molar ratio.
46. The composition of claim 45, wherein the cationic lipid constitutes about 40 % of the liposome by molar ratio.
47. The composition of any one of claims 40-46, wherein the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:30:20:10 by molar ratio.
48. The composition of any one of claims 40-46, wherein the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:30:25:5 by molar ratio.
49. The composition of any one of claims 40-46, wherein the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:32:25:3 by molar ratio.
50. The composition of any one of claims 38-50, wherein the liposome has a size less than about 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, or 50 nm.
51. The composition of any one of claims 38-50, wherein the composition is formulated for intravenous administration.
52. The composition of any one of claims 38-50, wherein the composition is formulated for pulmonary delivery.
53. The composition of claim 52, wherein the composition is formulated as respirable particles, nebulizable lipid, or inhalable dry powder.
54. The composition of any one of claims 38-50, wherein the composition is formulated for intrathecal administration.
55. The composition of any one of claims 38-54, wherein the mRNA has a length of or greater than about 0.5kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb.
56. The composition of any one of claims 38-55, wherein the protein encoded by the mRNA is Argininosuccinate Synthetase (ASS1), Factor IX, survival of motor neuron 1, or phenylalanine hydroxylase.
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